United States Environmental Monitoring Pre-lssuance Copy
Environmental Protection Systems Laboratory January, 1987
Agency P.O. Box 15027
Las Vegas NV 89114-5027
Research and Development
&EPA Carcinogen-DNA Adducts
Introduction,
Literature Summary,
and Recommendations
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CARCINOGEN-DNA ADDUCTS
INTRODUCTION, LITERATURE SUMMARY, AND RECOMMENDATIONS
by
S.D. Soileau
Environmental Programs
Lockheed-EMSCO, Inc.
Las Vegas, Nevada 89114
Contract No. 68-03-3249
Vork Assignment Manager
Tamar G. Gen
Exposure Assessment Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89114
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89114
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NOTICE
The information in this document has been funded wholly or in part by the
U.S. Environmental Protection Agency under contract number 68-03-3249 to
Lockheed Engineering and Management Services Company, Inc. It has been
subject to the Agency's peer and administrative review, and it has been
approved for publication as an Agency document.
Mention of trade names or commercial products doe6 not constitute
endorsement or recommendation for U6e.
ii
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ABSTRACT
This report summarizes the literature concerning adducts formed by
xenobiotics with DNA and protein in order to determine their feasibility as a
monitoring tool for use in exposure and risk assessment and to propose
compounds and methods that may be appropriate for preliminary field studies.
This report is divided into three segments.
The first segment provides an introduction to DNA damage and its relation
to carcinogenesis. This segment also discusses available methodology for the
measurement of macromolecular (DNA, protein) adducts. The techniques were
evaluated according to their sensitivity, selectivity, limitations, and future
possibilities. The next segment provides a summary of the current literature
on the individual chemicals found to form adducts in both man and in
experimental animals. The Information in this segment and additional
information was tabulated and is presented in the appendix. Finally, the
conclusion and recommendation section discusses the overall potential for the
use of macromolecular adducts as a measure of dose, given the current
technology. Recommendations on the analytical detection methodologies,
applicable chemicals, and populations to be used for a human monitoring pilot
study were offered.
ill
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CONTENTS
Abstract iii
Figures v
Tables vi
1. Introduction 1
2. MethodB for Detecting Carcinogen-DNA Adducts 17
3. Literature Review of Carcinogen DNA Adduct Structure,
Persistence, and Dose/Response Characteristics 23
4. Recommendations for Future Research 46
Appendix 53
References 100
iv
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FIGURES
Number Page
1 Sites of interaction of chemical carcinogens with DNA
in vivo (A) and i_n vitro (B). Adapted from (188a) 3
2 Examples of direct-acting carcinogens. Adapted from (188a). ... 4
3 Examples of indirect-acting carcinogens. Adapted from (188a). . . 5
4 Mispair^ng induced by O^-methylguanine.
A. 0 -Methylguanine-thymine base pair.
B. Normal G-C base pair. Adapted from (188a) 6
5 Relationship between initiation, promotion, and tumor formation
Adapted from (120) 8
6 Relationship between exposure, dose, and health effects.
Adapted from (220a) 12
v
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TABLES
Number Page
1 Protein Binding of Alkylating Agents 14
2 Assays for the Detection of Carcinogen DNA Adducte 16
3 Co^ounds Tested for DNA Binding in vivo by
P-postlabeling analysis 20
vi
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INTRODUCTION
The Environmental Protection Agency is charged to protect human health
and the environment, and It has acted by placing restrictions and regulations
on chemicals that have been shown to be detrimental to human health or to the
environment. Accurate dose measurements are critical in the evaluation of
health risks and in the development of regulations that may be needed for
protection from chemicals released Into the environment.
In the past, human exposure to xenoblotics has been estimated by direct
measurement of the concentration or amount of the xenoblotic present in one or
more environmental compartments (e.g., air, water, foodstuffs, etc.). Such
data can only give crude estimates of the dose received because additional
information is required to estimate more accurately the dose (e.g., duration
of exposure, pulmonary ventilation, food consumption, etc.).
Biological monitoring is the measurement of the concentration of
xenoblotics in organisms (e.g., man). Examples of biological monitoring would
Include measurement of xenoblotics or their metabolites in blood or urine or
measurement of reaction products between the compounds and cellular
macromolecules such as proteins and ONA. Biological monitoring gives a better
estimate of the dose received because it corrects for interindividual
variations in absorption, metabolism, and excretion. This kind of chemical
dosimetry also integrates exposure from all sources, and therefore can be used
as a basis for the estimation of the total potential risks from multiple
chemicals. Because of these correcting factors, biomonitoring is related more
directly than environmental measurement to the adverse effects induced by
xenobiotics (41).
The Environmental Protection Agency has developed an initiative designed
to develop, refine, and apply appropriate biomarkers that can be used in
conjunction with other environmental monitoring data to provide a better
estimate of risk to individuals and populations. By linking biological
measurements to environmental monitoring measurements, it will be possible to
determine relationships that exist between total exposure, dose, and disease.
The first stage of the EPA initiative is to evaluate the feasibility of
using biomarkers as a monitoring tool for use in exposure and risk assessment.
This will Include a compilation of available biomonitoring methods for
assessing environmental exposures and of methods for predicting associated
health risks. The purpose of this document is to summarize the literature
concerning adducts formed by xenoblotics with DNA and protein in order to
determine their feasibility for use In exposure and risk assessment and to
propose compounds and methods that may be appropriate for preliminary field
studies. This report is divided into three segments.
The first segment provides an introduction to DNA damage and Its relation
to carcinogenesis. This segment also discusses available methodology for the
measurement of macromolecular (DNA, protein) adducts. The techniques were
evaluated according to their sensitivity, selectivity, limitations, and future
possibilities. The next segment provides a summary of the current literature
on the individual chemicals found to form adducts In both man and experimental
1
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animals. The Information in this segment was also tabulated and is located in
the appendix. Finally, the conclusion and recommendation section discusses
the overall potential for the use of macromolecular adducts as a measure of
dose, given the current technology. Recommendations on the analytical
detection methodologies, applicable chemicals, and populations to be used for
a human monitoring pilot study were suggested.
Chemical Carcinogenesis
The ability of chemicals to induce cancer has been known for more than two
centuries. It was observed in 1776 by Sir Percival Pott that chimney sweeps
developed scrotal cancer and that the cancer was associated with exposure to
soot and tars. Initially, it was believed that chronic Irritation was the
cause of cancer, but this theory could not explain how short exposures to
chemicals were sufficient to induce cancer and that cancer could appear many
years after exposure to carcinogenic chemicals. Many chemicals were found to
be carcinogenic, but there appeared to be no correlation between chemical
structure and the ability of a particular chemical to induce cancer.
Researchers found that the metabolites of some carcinogens were more active
than the parent compounds themselves. They then looked at the compounds and
their active metabolites to determine if similarities existed in their
chemical activities. It was discovered that the great majority of active
compounds contained an electrophillic group (i.e., an atom possessing a low
electron density). These electron-poor sites attack sites of high electron
density. Figure 1 shows sites of Interaction of chemical carcinogens with DNA
in vivo and in vitro. Nitrogen and oxygen atoms in proteins are also
susceptible to attack.
These electrophillic compounds can be divided into two classes; direct
acting and indirect acting carcinogens. Direct acting compounds possess
strong electrophillic sites and can covalently bind to DNA without chemical
modification. Nitroso compounds are an example of direct acting carcinogens.
Figure 2 shows other carcinogens of this class. Indirect acting carcinogens
cannot alkylate DNA directly; they must be metabolically activated to form an
electrophillic species before alkylatlon can occur. Benzo(a)pyrene is an
example of an indirect acting carcinogen. It must be metabolically activated
to form the diol-epoxide metabolite before alkylatlon can occur. Figure 3
shows some other carcinogens of this class. It was postulated that the
compounds of Interest may damage DNA and that this damage was the first step
in chemical carcinogenesis. It was also postulated that this damage muBt be
inherited by daughter cells, so the damage must induce a change In the DNA
that can be passed on to future generations of cells. In short, a mutational
change in the DNA sequence within a gene must be induced in the affected cell.
Mutation induced by DNA alkylatlon is postulated to occur by the following
mechanism. The active form of the carcinogen alkylates DNA in such a way as
to alter the manner in which i£ base pairs during replication. An example
would be the base pairing of 0 -methyl guanine. Guanine usually base pairs
with cytosine (Figure 4). However, if guanine is methylated at the 06
position, its ability to hydrogen bond is altered to the extent that it
prefers to base pair with thymine. When replication of the single strand of
DNA containing the methylated guanine occurs, a thymine will replace cytosine
at the position of the alkylated guanine (Figure 4). Therefore, alkylatlon of
bases in DNA can induce the formation of stable mutations. These changes in
the DNA sequence can result in failure to transmit genetic information
accurately.
2
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MNU
3,4 BENZPYRENE ?
7 BrMBA
DNA CHAIN
'8CH
DNA CHAIN
DMN; MNU; MMS; MNNQ
MNU; DMN; MMS; MNNQ
URETHAN ?
7BrMBA
MMS: DMS; MNNQ: DMN-
H 1 H
"hVm
^ Ml . I
DMN; DEN; MNU; ENll; MNNQ; SAFROLE
DNA CHAIN
B
MNU; DMS
" \ t
I MNU;
rscH-
DNA CHAIN
DMS
DMN; DEN; ENU; MNNQ;
MAM; MMS; EMS: ETHIONINE
1,2 DMH: BIFUNCTIONAL ALKYLATING
AGENTS;BPL
2AAF; MAB?; 4NOQ7; 7BrMBA
7BrMBA
Figure 1. Sites of interaction of chemicai carcinogens with DMA
in vivo (A) and in vitro (B). Adapted from (188a).
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ALKYLATING AGENTS
ch2
I
o
ch2
I
c.
ch3-o^ o
s
CH3-O ^
B-PROPIO LACTONE
DIMETHYLSULFATE
H3N,
CI
pi n
H,N'
'CI
CISPLATIN
CH2
/ \
ch2 ch2
\ /
o — s —* o
I
o
PROPANE SULTONE
/°\
ETHYLENE OXIDE
^nh-ch2-ch2ci
0= C
ch2 ch2 ^n-ch2-ch2ci
V.
/
0= N
BCNU
Figure 2. Examples of direct-acting carcinogens. Adapted from (188a).
4
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oTo
oToTo
CH;
o
ololo
BENZ(a)PYRENE
CH3
7,12-DIMETHYLBEN2CaJ-
ANTHRACENE
C-CHg
2-ACETYLAMINOFLUORENE
N-METHYU-4-AMJNO-
AZOBENZENE
CH3
no2
4-NtTROQUINOLINE- 1-OXIDE
c2hs-o-c
NH.
URETHAN
C2H5.
N-NO
C2H5'
OIETHYLNITROSAMINE
C2Ha-S-CH2-CH2-CH-COOH
NH2
ETHIONINE
Figure 3, Examples of indirect-acting carcinogens. Adapted from (18Ba).
5
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OCH
Figure 4. Mispairlng Induced by Oft-methylguanlne.
A. o'-Methylguanine-thymine base pair.
B. Normal G-C base pair. Adapted from (188a).
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However, genotoxlc damage, In and of itself, la not sufficient to induce
tumor formation. In 1941, Rous and Kidd first suggested a two stage mechanism
for the development of cancer: an initiation step followed by promotion with
another agent (186). Initiation is essentially the damage of cellular DNA
that results in DNA miscoding. The term promotion is used to designate the
process by which initiated cells are encouraged or accelerated to become
neoplastic (i.e., cancerous). This hypothesis was extended by Berenblum and
Shubik in the late 1940's (36). The investigators determined that neither
methylcholanthrene (the initiator) nor the croton oil (the promotor) produced
tumors when applied separately. However, when the promoter was applied after
application of the initiator, tumor development was noted (Figure 5).
Animal experiments indicate that initiation is an irreversible event,
whereas promotion is reversible. Application of a promotor to an experimental
animal that was treated with an Initiator a year previously still resulted in
skin tumor formation (220). However, if application of a promotor is
discontinued before the cells are transformed to a neoplastic state, tumor
development is not seen.
Genotoxic Damage and its Relattonshlp to Carcinogenesis/Hutagenesia
Although initiation implies DNA damage, not all DNA damage can be termed
initiation as such. If DNA damage is very severe, the cell Is unable to
produce essential proteins and the cell dies. Sister chromatid exchange (SCE)
is a type of chromosomal damage that is characterized by the transfer of
chromatin between two chromatids of a chromosome. Although cigarette smoke
condensate can Induce a concentration-dependent increase in the frequency of
SCE's in human lymphocytes (95), SCE's appear to correlate better with cell
death than with cell mutation rates (36,152). DNA damage can also be
expressed as actual breaks in the DNA strand. Another gross change that has
been used as a method of detecting DNA damage is the measurement of
micronuclei in maturing erythrocytes and in lymphocytes. Colchicine, a drug
that inhibits mitotic spindle formation, can induce micronuclei formation in
dividing cells. Unscheduled DNA synthesis (UDS) is also a measure of DNA
damage. UDS is a measure of excision repair of damaged DNA. This means that
damaged DNA is removed enzymatically and is replaced with undamaged
nucleotides.
Although a mutation is postulated to be required in the multistage process
of cancer induction, not all mutations will lead to the induction of cancer.
For example, a mutation may lead to the change in a single amino acid in a
particular protein or enzyme. This may or may not be fatal, but it is not a
cancer initiation step. Congenital enzyme deficiencies, such as
Phenylketonuria (PKU), or Sickle Cell Anemia are examples of this type of
mutation. A mutation may occur such that an amino acid codon is converted to
a stop codon. This type of mutation is usually fatal. Blfunctional
alkylating agents can cause crosslinking of double-stranded DNA and can
prevent complete DNA replication and cause cell death if not repaired. Many
cancer chemotherapeutic agents, such as cisplatin, cause cell death via such a
mechanism. It Is not known how mutations cause normal cells to become
neoplastic, but there is evidence that some mutations activate certain genes
that can transform a cell to a neoplastic state (205,210). These genes are
referred to as oncogenes. Experimental evidence indicates that chemical
carcinogens, in concert with certain viruses, can trigger the neoplastic
transformation of human epithelial cells (183a). The oncogenes may be present
7
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methylcholanthrene
(initiator)
no tumor
no tumor
skin tumor
croton oil
(promoter)
methylcholanthrene
(initiator)
croton oil
(promoter)
5. Relationship between initiation,
Adapted from (120).
8
promotion,
and tumor formation.
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In the cellular genome, or they may be Introduced ioto the cell by means of a
virus. In summary, very specific mutations are required for neoplastic
conversions to occur. Although very specific mutations are required for
neoplastic conversion, non neoplastic-inducing mutations also take place.
Biological methods exist for both in vivo and in vitro measurement of the
Induction of mutations. The Ames test measures the ability of a chemical to
induce a specific mutation in the his Salmonella bacteria to allow the mutant
bacteria to synthesize histidine. Other assays measure hypoxanthine guanine
phosphorlbosyl transferase (HGPRT) and hemoglobin mutations in blood cells.
It is believed that the primary mechanism of chemically-induced mutation
is through miscoding induced by carcinogen-DNA adducts. The existence of ONA
adducts was first shown in 1962; a specific alkylated base, 7-methylguanine,
was isolated from cells treated with dimethylnitrosamine (137). Even though
this adduct is now considered not to be important in the mechanism of
carcinogenicity of alkylating agents, this study was important in that it
stimulated a large number of studies in the area of carcinogen-DNA adducts.
Many studies have shown that carcinogen-DNA adduct levels are correlated
with the frequency of mutagenic/carcinogenic alterations. Alkylating agents
can attack nitrogen and oxygen atoms in purines and pyrimidines in DNA, agd in
many instances, a relationship was found to exist between the levels of 0 -
alkylguanlne and the frequency of tumor occurrence (148, 208, 222). Most
other carcinogens show a similar correlation. 2-Napthylamine (2-NA) is a
urinary bladder carcinogen but not a liver carcinogen in dogs. The binding of
radiolabeled 2-NA to DNA In dog bladder and liver was measured in a recent
study. Eight days after administration of 2-NA, total binding to DNA was
eight times higher in the bladder than in the liver (106). In another study
1-napthylamine (1-NA) and 2-NA were fed to Sprague-Dawley rats. 1-NA was
found to be more carcinogenic than 2-NA, and this difference was correlated
with the greater binding (about 20 fold) of 1-NA to DNA (60).
Unfortunately, the relationship between DNA adduct levels and the
frequency of tumor formation is rarely a simple direct correlation. For
example, when mice were treated with the carcinogen 15, 16-dihydro-ll-
methylcyclopenta[a]phenanthrene, initial DNA levels in mouse liver were twice
as high as were found in skin and lung DNA (2). However, this compound Is
carcinogenic in mouse lung and skin but not in the liver. Vhen the
persistence of the adducts was measured, it was found that although liver DNA
contained higher initial levels of DNA adducts, repair of the damage occurred
at a faster rate than in mouse lung or skin (2). It therefore appears that
the persistence of DNA adducts is an important factor in relating DNA adduct
levels to tumor frequency. In several other studies, the same inverse
correlation was found between DNA repair of chemically induced lesions and the
frequency of mutagenic or carcinogenic events. 7, 12-Dimethylbenz[a]-
anthracene, a potent mammary carcinogen in certain strains of rats, was
administered to a resistant strain of rat, the Long-Evans rat, and to a
susceptible strain, the Sprague-Dawley rat (50). Initial DNA adduct levels
were significantly higher in the resistant strain. However, the resistant
strain showed significant DNA repair 14 days after carcinogen administration,
whereas the susceptible strain showed no significant loss of DNA addicts.
Using genetic engineering techniques, a tetranucleotide containing 0 -methy1-
guanlne was spliced into an E. coll bacteriophage. The virus (bacteriophage)
was allowed to infect cells with either a normal or deficient DNA repair
mechanism. Because viruses use host cell enzymes for their own replication,
9
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one would expect to find an increased mutation frequency In the progeny virus
from the repair-deficient cells. An increased mutation frequency was found;
in fact, the mutation frequency was 50 times higher in the repair-deficient
progeny phage (62). A study that measured the formation of aflatoxln Bj In
rat liver showed that the initial principal adduct was removed with a halfllfe
of 6.5 hours. However, a small percentage (20 percent) of the adducted
guanine residues underwent an imidazole ring scission, and these ring-opened
adducts were not rapidly repaired. If multiple doses were administered in a
regimen shown to produce a high incidence of hepatocellular carcinoma, a time-
dependent increase in the imidazole ring-opened adduct was detected (48).
Similarly, the persistence of 3-methylcholanthrene (3-MC) binding in mouse
liver and lung DNA was measured for up to 28 days after administration of a
single i.v. dose. Mouse lung is susceptible to 3-MC (in terms of
carcinogenicity), whereas mouse liver Is resistant. Both tissues showed a
decrease in adduct levels 28 days after administration of 3-MC, however,
adduct levels were still measurable in lung DNA, whereas adduct levels in the
liver were no longer measurable (61a).
Another factor that must be considered when relating adduct levels to
carcinogenesis is the rate of cell division of the damaged cell. Unless the
damaged nucleotides are 'fixed' by miscoding during replication before DNA
repair occurs, a mutational event will not occur. The relatively high rate of
cell division in mouse lung and skin cells may partially explain why such
cells are susceptible to 15, 16-dihydro-ll-methylcyclopenta[a]phenanthrene-17-
one, even though initial adduct levels in the liver are twice as high (62).
The rate of cell division in the liver is normally quite low. A recent report
by Swenberg et al. (208) proposed that all promutagenic ONA adducts are
important in the mechanism of carcinogenesis, and the extent of cell
replication for each population of cells exposed influences the probability of
tumor formation.
These three factors (adduct levels, adduct repair, and rate of cell
division) are Important in relating DNA adducts to carcinogenesis, but other
unknown factors are probably important. DNA adducts were found to be
persistent in rat liver not only for two hepatocarcinogens, N-hydroxy-2-
acetylaminofluorene (N-OH-AAF) and N-hydroxy-4-acetylaminoblphenyl, but also
for the non-hepatocarcinogen, N-hydroxy-2-acetylamlnophenanthrene (75). In
another study, persistence of DNA adducts in a non-target tissue (kidney) was
noted after multiple treatments with N-OH-AAF (31). In summary, although a
correlation has been noted between certain DNA adducts and tumor formation,
research is still needed to elucidate all of the mechanisms involved In the
carcinogenic process.
As has been discussed earlier, exposure assessment la accomplished at
present by monitoring the environment for carcinogens and also by monitoring
biological fluids or tissues for the same. Monitoring biological fluids or
tissues for carcinogens Is more accurate than environmental data because it
factors In the effects of variation in absorption and metabolism of the the
compounds. However, measurement of free carcinogen concentrations does not
measure actual DNA damage or related phenomena (i.e., protein adduct
formation). For example, interlndlvidual variations in binding of
benzo[a]pyrene to DNA is about 50- to 200-fold (218, 78). Other compounds
show at least 10-fold variation among individuals (218). Measurement of
damage in the organ that is susceptible to the carcinogen (target site) would
provide a more accurate estimate of the risk (17). Carcinogen adducts would
10
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provide such a measure of the dose received. DNA adducts give a measure of
genotoxlc exposure, because the levels measured are the net of the adducts
formed minus adducts lost through enzymatic or non-enzymatlc repair,or both.
Protein adducts, on the other hand, are stable over the life span of the
protein and as such provide an integration of exposure over the life of the
protein (235). Because of these properties, measurement of DNA adduct levels
could be more useful in risk assessment, and measurement of protein adduct
levels could be more useful in exposure assessment.
Biological Markers for DNA Damage
llhen an organism Is exposed to a genotoxlc agent, many sites are attacked.
DNA of both susceptible and resistant cell types are attacked, and other
molecules containing nucleophilllc sites, such as proteins and RNA, can also
be modified by the same electrophlllic agents. Because of the non-specific
action of electrophiles, there are many types of molecules that can be used as
molecular dosimeters for genotoxlc damage.
The ideal dosimeter for relating dose to biological effect Is the DNA from
the cell populatlon(s) that is susceptible to the particular carcinogen being
studied. Different cell types have different levels of xenobiotic-
metabollzing enzymes and DNA repair enzymes, and physical properties of the
carcinogen may result in different levels of the carcinogen in different cell
types. There are many steps between exposure to a carcinogen and the
Induction of target cell DNA damage (Figure 6). Small changes in any or all
of these steps can lead to great changes In the levels of DNA adducts. For
these reasons, measuring DNA adduct levels In the target tissue would give the
best possible correlation between environmental levels of the carcinogen and
target site damage, as well as the best possible correlation between target
site damage and tumor formation. Unfortunately, there is an important
disadvantage to using target site DNA as a molecular dosimeter: accessibility.
It is extremely difficult to obtain a sample; a biopsy, at a minimum, is
usually required. Samples of this type are usually obtained during elective
surgery or during an autopsy conducted immediately after death. Even though
difficult to obtain, such samples can be of some use. For example,
benzo[a]pyrene-DNA adducts were detected in cultured human colon of persons
with and without colon cancer (13). The, use of this sample in an epidemiology
study with a large number of participants is essentially impossible.
The problem of availability can be averted by measuring DNA adduct levels
in tissues and fluids that are easily accessible and that can be obtained with
a minimum of invasiveness. Circulating DNA, in the form of white blood cell
(UBC) DNA, is readily available and would be amenable for use in a large
epidemiology study. The main disadvantage of this sample type Is that the
levels of DNA adducts found in WBC's may not relate to the levels of DNA
adducts at the target site.
DNA damage can also be measured indirectly by measuring DNA adducts that
have been excreted in urine. The detection of DNA adducts in urine would
theoretically give an Indication of recent exposure to certain compounds. In
addition, If Information were available on the dose received, such
measurements would indicate the level of DNA repair in the individual (235).
For a given dose received, the more rapid the repair, the more adducts would
be detected in the urine. This information would complement DNA adduct level
data measured in either the target or circulating DNA. Aflatoxln Bl-DNA
11
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ENVIRONMENTAL METABOLIC
LEVELS OF UPTAKE PRECURSOR DEACTIVATION
PRECURSOR CARCINOGEN
CARCINOGEN
ACTIVATION:
(1) SPONTANEOUS
(2) MICROSOMAL
OXYGENASE
(3) OXIDATION AND
CONJUGATION
DIRECT-ACTING
CARCINOGEN
OR MUTAGEN
RNA AODUCTS
PROTEIN ADDUCTS
Figure 6 Relationship between exposure, dose
from (220a)
EXCRETION
(URINE. FECES)
DEACTIVATED
METABOLITE
DEACTIVATION, E.G.,
(1)SPONTANEOUS
(2) GLUTATHIONE-S-
TRANSFERASE
(3) EPOXIDE HYDRATASE
ULTIMATE CARCINOGEN
OR MUTAGEN
COVALENT BINDING
WITH CELLULAR
MACROMOLECULES
DNA ADDUCTS
REPAIR
ALKYLATED
NUCLEIC
ACID BASES
CELLULAR LESIONS
TIME
TUMOR
and health effects. Adapted
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adduct excretion.has been monitored by using this strategy (15, 35). The
disadvantage of this method is that measurement of excreted DNA adducts gives
no indication as to the cell population(s) that contained the adducts. For
this source to be a useful indicator of dose, studies would have to be
conducted to understand the relationship between adduct levels in the urine
and adduct levels in the target cell DNA.
If one wants to determine ONA adduct structure or if one wants to study
reaction mechanisms of the compounds with DNA, in vitro studies can be of some
use. The compounds of interest are reacted with DNA (usually calf thymus DNA)
and isolation, characterization, and quantitation of the carcinogen-DNA
adducts are performed in a manner similar to that used in in vivo studies.
Several compounds have been studied in this manner, Including derivatives of
benzo[a]pyrene (72), azo dye derivatives (135, 213) and 15, 16-dihydro-ll-
methylcyclo-penta[a]phenanthren-17-one (47). This procedure offers the
advantage of greatly increasing the levels of adducts in the DNA, thus
facilitating identification of adduct structure. If the carcinogen is direct
acting, that is, it requires no metabolic activation, the compound is simply
mixed with the DNA in an aqueous solution. If the compound requires metabolic
activation, either an enzymatic metabolizing system must be Included (47), or
a stable derivative of the electrophilic compound must be synthesized (22,
135, 213). One disadvantage of this method is that the adducts formed may not
be the same as the ones formed l_n vivo (22).
Although measurements of DNA adducts are an ideal measurement when one
wants to relate environmental levels to biological endpoints, they are not
necessarily an ideal measure of dose. The levels of DNA adducts are modulated
by enzymatic and nonenzymatic repair, and cell division can dilute the
concentration of the adducts. As was stated earlier, electrophillic compounds
also form covalent adducts with proteins. Carcinogen-protein adducts are
usually stable over the life of the protein and as such make a good integrator
of exposure during this time period. Most of the early studies have measured
binding to proteins by using radiolabeled carcinogens, although GC-MS or
amino acid sequencing techniques have also been used (64, 99; Table 1). These
adducts should be easily amenable to immunological methods of detection, and
detection levels should be very low. Hemoglobin has received the most
attention in this area, presumably because it is easily accessible and because
it is possible to obtain large quantities of the protein. The lifespan of an
erythrocyte is about 120 days, so measurement of hemoglobin adducts integrates
exposure over months. Human serum albumin has a half life of about 20 days
(232). Protein adducts measured on serum albumin would integrate exposure
over a much shorter time span. Protein adduct formation shows a linear dose-
response curve for most compounds (20, 64, 99, 155, 164, 193, 212), and
protein adduct formation tends to correlate with DNA adduct formation in
various tissues. If one wanted to correlate a particular DNA adduct formation
with protein adduct formation, the appropriate animal experiments would have
to be conducted. Because the levels of adducts that would be detected in the
general population would be very low, one would have to make sure that the
dose response relationship was linear at very low carcinogen concentrations.
One would not expect the correlation between DNA and protein adduct levels to
be perfect in continuous dosing regimen because of the occurrence of DNA
repair, cell replication, etc. Also, the toxicokinetics of the carcinogen may
also affect DNA adduct formation in a different manner.
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TABLE 1. PROTEIN BINDING OF ALKYLATING AGENTS
COMPOUND
PROTEIN
SPECIES
Methyl methanesulfonate
Hb
mouse
Hb
rat
N-Nltrosodimethylamine
Hb
mouse
Serum
rat
Hb
rat
Erythrocyte
human
Methyl bromide
Hb
mouse
Methyl chloride
Erythrocyte
human
plasma
human
Dichlorvos
Hb
mouse
Ethylene oxide
Hb
mouse
Hb
human
Hb
rat
Propylene oxide
Hb
rat
Vinyl chloride
Hb
mouse
Ethylene
Hb
mouse
Benzo[a]pyrene
Hb
mouse
Hb
rat
Chloroform
Hb
rat
Hb
mouse
2-Acetylaminofluorene
Hb
rat
Hb
mouse
Serum
rat
Benzyl chloride
Hb
mouse
Aflatoxin BI
Hb
rat
trans-Dimethylaminostilbene
Hb
rat
Plasma
rat
trans-4-Aminostilbene
Hb
rat
Plasma
rat
4-Aminobiphenyl
Hb
rat
Albumin
rat
Hb = Hemoglobin
NOTE: Adapted from (64)
14
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Although it is possible to measure both DNA and protein adducts in humans,
it will likely be more difficult to relate ONA adduct levels to environmental
levels. As can be seen In Figure 6, many factors influence the level of an
electrophillic compound in an organism (or humans). If one wants to relate
environmental levels of carcinogens to levels in the body, one has to correct
for inter individual variation. A direct or indirect approach can be used for
this correction. In the direct approach, one could try to define all of the
factors that influence levels of a compound in the body and then try to
quantitate each factor for each individual in the study. This would involve,
at a minimum, measurement of various enzyme levels. Even if all factors could
be measured quantitatively, this model would assume that enzyme levels remain
constant. This is certainly not the case. Additional animal experimentation
would be required for this approach to be viable. One idea that might be
investigated is to determine if the repair capability of an individual can be
estimated by determining the ratio of DNA or protein adducts in the measured
tissue or fluid to the levels of DNA adducts in the urine. It might also be
helpful to measure levels of metabolites in the urine. This would given an
indication as to how much detoxification of electrophillic species occurred.
In the indirect approach, a large enough sample size is chosen such that
inter individual variation tends to cancel out. Another problem that must be
faced is the randomness of dosing in the real world. In the general
population, dosing would not occur as a single dose of a carcinogen; rather,
small doses of many carcinogens would occur at irregular time Intervals. It
is likely that many preliminary animal experiments would be required in order
to understand human dose-response relationships in the general population.
Even though much research still needs to be conducted, the measurement of
adduct levels in individuals would provide a much better estimate of dose than
the measurement of environmental levels of carcinogens.
15
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TABLE 2. ASSAYS FOR THE DETECTION OF CARCINOGEN-ONA ADOUCTS
METHOD
Limit
Of
detection
fmole
Amount of DNA used
Modificatio
analyzed 0N>
per analysis mo adducts/base
UV in line with HPLC
[major benzo(a)pyrene-ONA adduct ]
100,000
2600
2 x 10"1
Fluorescence in line with HPLC
(BPOE-I-tetrol)
31
100
1 * 10~~
Photon counting synchronous scanning fluorimetry
Imnunoassays
Polyclonal rabbit antibodies against
BPDE-I-ONA
1 x 10-
Competitive assays
RIA
ELISA
USERIA
USERIA
5,300
55
12
10
1
1
1
25
1.7 x 10"1
1.8 x 10~—
3.9 x 10"-
1.4 x 10"-
Noncompetitive assays
USERIA
0.01
9.7 x 10"-
Honoclonal antibodies against BPDE-I-ONA
Competitive ELISA
Noncompetitive ELISA
19
3
0.005
0.0002
1.2 x 10"1
4.9 x 10"—
12
P-postlabeling
0.03-0.3
1
lxl0~--10~-
As a guideline, 10-100 mg DNA is recoverable from 0.2-1 g tissue or the buffy coat of 25-50 ml hunan blood.
Adapted from (235).
Benzo(a)pyrene is used as an example in this table since these techniques have been applied to the detection
of its major adduct, 10-(deoxyguanosin-M2'-y1)-7l8l9-trihydroxy-7,8t9,10-tetrahydrobenzo(a)pyrene.
16
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METHODS FOR DETECTING CARCINOGEN-DNA ADDUCTS
During the past 10 years, many different methods have been developed to
measure DNA-carcinogen adducts. The methods have widely varying sensitivities
and varying levels of applicability. Table 2 lists available methods,
sensitivities, and amount of DNA required for analysis. In the discussion of
each of the methods, isolation of the DNA from the selected sample is required
prior to the use of the detection methods.
Most of the work in DNA adduct research in animal models has been
accomplished using radiolabeled carcinogens and subsequent measurement o^the
radioactivity via scintillation counting. The primary isotopes used are C
and H. The advantage of H as the isotope is that it is usually easily
incorporated into compounds and can be obtained at a relatively high specific
activity. The use of a high specifi^ activity radioisotope increases the
sensitivity of the compound. ^|ing H, adducts can be detected down to |
level of a few femtomoles (10 moles) of adduct. The disadvantage of H in
animal studies Is that It is susceptible to loss during metabolism or by
exchange re^tions. These reactions can lead to misinterpretation of results.
The use of C will circumvent exchange and metabolism problems, but the
detection limit is about 200 times higher than that achieved with H (101).
Because the general public is not exposed to radiolabeled carcinogens, this
method would not be useful in epidemiological studies.
High performance liquid chromatography coupled with an ultraviolet
absorbance det|Cjtor has a limit of detection in the range of hundreds of
picomoles (10 moles) of adduct. This method is useful for many adducts
because many carcinogens show strong absorbance at 254 nm, the usual
wavelength of the detector. Although this procedure could be used in the
study of DNA adduct formation in humans, it is usually used to measure adducts
obtained from experimental animals because the method is insensitive relative
to other methods. Examples of DNA adducts that can be measured by using this
technique are polynuclear aromatic hydrocarbons and benzidine derivatives.
Similarly, because polycyclic aromatic hydrocarbons are highly fluorescent
molecules, fluorescence has been used to measure DNA adducts (219). The main
disadvantage of this detection method is that it is relatively insensitive.
Improvements in sensitivity were achieved by using a method called synchronous
fluoresence spectrophotometry. This involves holding the difference between
the excitation and emission wavelengths constant and scanning across the
ultraviolet and visible spectrum. Detection limits as low as 31 femtomoles
(10 moles) have been achieved with benzo(a)pyrene (219). Although this
method is sensitive, is suffers from its lack of universality. It is only
useful when studying compounds that fluoresce, and this property is primarily
limited to polycyclic aromatic hydrocarbons.
Chemical derivatlzation coupled with gas chromatography/electron capture
detection is also a viable detection technique. In this procedure, the DNA is
broken down to nucleotides, and the nucleotides are derivatlzed to increase
nucleotide volatility and to Increase detector sensitivity. In a recent
study, cytoslne was derivatlzed with pentafluorbenzoyl chloride and dimethyl
sulfate (66). As little as 50 nmol of starting material (cytoslne standard)
can be detected. Although this procedure shows some promise, it remains to be
seen how well the derivatlzation procedure will work using 'real world'
samples; the derivatlzation step may create too many interferences. It would
17
-------
be a convenient technique because of the wide availability of gas
chromatographs.
Much literature has been published in the area of antibodies to
carcinogen-DMA adducts. The determination of carcinogen-DNA adducts by
immunologic procedures has certain advantages over other techniques. The
sensitivity is frequently better than that obtainable with radiolabeled
carcinogens. Antibodies are very selective for a particular three-dimensional
structure and as such show very little or no cross reactivity with similar
compounds. Immunologic assays are rapid, are highly reproducible, and can be
used in situations where the cost of a radiolabeled compound would be
prohibitive. Because one can measure nonradioactive DNA adducts, the
procedure would lend Itself to use in monitoring human tissues (174).
Antibodies to particular adducts are usually prepared by covalently
binding the adduct of interest to a carrier protein or by using DNA that was
modified in vitro. This is done because the size of a purine or pyrimidlne
base adduct is not large enough to stimulate an antigenic response. The
protein-or DNA-adduct moiety is injected into an appropriate animal: rabbit,
mouse, rat, etc. After a few months and several injections, the antibody
titer is at maximal level (174). This is the general way in which polyclonal
antibodies are prepared. As the name implies, the serum contains more than
one clone of antibodies; there are thousands or even millions. Since each
antibody has its own three-dimensional recognition site, the selectivity of
polyclonal antibodies may or may not be exceptional.
If one could isolate only one antibody clone, one would expect to obtain
maximal selectivity. This is the rationale behind the preparation of
-monoclonal antibodies. A preparation of monoclonal antibodies contains only
one antibody clone. The antibodies are prepared by using hybridoma
technology. Animals are innoculated with the antigen of choice in a manner
identical to that used in polyclonal antibody production. The animals are
sacrificed, and the spleen cells are removed. The spleen cells are fused with
mutant myeloma cells under conditions such that only fused cells are viable.
The fusion creates cells that can be cultured indefinitely. The antibodies
with the desired specificity and sensitivity can then be located and selected.
The spleen is a rich source of B cells, and It is B cells that produce
antibodies. Therefore, each fusion that occurs between a B cell and a myeloma
cell creates a cell that is immortal and produces a unique antibody. The main
advantage of monoclonal antibodies is that they can be very selective.
However, monoclonal antibodies do not necessarily have to be selective; for
example, if a monoclonal antibody binds to a chemical site that Is common to
chemicals of a particular class (e.g., the hydroxyl portion of chlorinated
phenols), selectivity would be poor. The main disadvantages of monoclonal
antibodies are that they are expensive to produce, they are time-consuming to
prepare, and luck plays a large part in locating an antibody with the desired
specificity and sensitivity. Once the appropriate antibody is located, the
assay is a very economical monitoring tool.
Polyclonal and monoclonal antibodies are used in various immunoassays:
radioimmunoassay (RIA) including the radioimmunosorbent technique (RIST),
enzyme-linked immunosorbent assays (ELISA), and ultrasensitive enzymatic
radioimmunoassays (USERIA). All of these techniques could be used to monitor
DNA adduct levels in human tissues.
18
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RIA is a competitive assay where two identical haptens compete for the
same antibody binding site. A hapten is a small molecule that cair bind to an
antibody but cannot by itself elicit an immunogenic response. One hapten, of
known concentration and radiolabeled, is added to a fixed amount of antibody.
The sample, containing an unknown quantity of hapten, is added, and the
solution is allowed to attain equilibrium. The amount of radioactivity bound
to the antibody will be inversely proportional to the amount of unknown hapten
present. After equilibrium is reached, the antibody-hapten complex is
precipitated. The amount of radioactivity is quantitated, and the level of
unlabelled hapten is calculated by using a standard curve. The limit of
detection in this assay is 5300 fmole of adducts or about one ONA adduct per
600 bases (benzo[a]pyrene; Table 2). Other examples of DNA adducts that have
been quantitated by this method include several alkylated guanosines (153,
188, 233) and adducts from the carcinogen 2-acetylaminofluorene (176).
ELISA, as the name implies, uses an enzyme to measure the level of binding
of a particular antibody to an antigen bound to a solid support (i.e., a
microtlter plate). This is also a competitive assay. Essentially, an
antibody is Incubated in an antigen coated well in the presence of added
antigen (ONA adduct sample).
The antibody is allowed to equilibrate between the free antigen in the
solution and the antigen bound to the well. After equilibration, the well is
washed, and a secondary antibody is added to the well. This antibody is
specific for the primary antibody, and it binds to the bound antibody. The
second antibody is conjugated to an enzyme that can react with an uncolored
substrate to create a colored product. The free second antibody is washed
out, and a substrate solution is added. The wells are incubated for a short
period of time, and the enzymatically produced color is measured. The
concentration of the added hapten can be calculated by using a standard curve.
The limit of detection of this method in treasuring benzo(a)pyrene-DNA adducts
is 55 fmol or about one adduct in 5 x 10 bases (235). Alkylated guanosines
(153) and aflatoxin-DNA adducts (72, 92) have also been measured by using this
technique. The reason this procedure is so much more sensitive is that two
amplification steps have been added. The first amplification arises from the
fact that several secondary antibodies bind to each primary antibody, and the
second amplification occurs because each enzyme molecule can convert many
substrate molecules into colored products.
USERIA is a technique that is essentially Identical to the ELISA technique
except that the enzyme substrate is radiolabeled. The enzyme converts the
radioactive substrate into a radioactive product. The product is chromato-
graphically separated from the substrate, and the product is quantified. The
detection limit for benzo(a)pyrene is about 10 fmole or about one adduct in 7
x 10" bases. USERIA has been used to quantitate several different types of
adducts (72, 92, 97, 153).
RIST is very similar to ELISA and USERIA; the difference lies in the fact
that the secondary antibody is radiolabeled. After the secondary antibody Is
bound to the primary antibody, the plates are washed, and the bound radio-
activity 18 counted. The sensitivity of this procedure is about the same as
the ELISA (153).
In summary, immunoassays possess many advantages; native, not digested,
DNA is used for analyses, the procedure is quick and inexpensive, many samples
19
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TABLE 3. COMPOUNDS TESTED FOR DNA BINDING
In vivo BY 32P-POSTLABELING ANALYSIS
QM arifluctf?
COMPOUND
TISSUE
No.b
Levels
Arylamlnes and derivatives
2-Acetylaminofluorene
MS
6
++
RL
11
+++
4-Acetylaminofluorene
RL
2
+
N-Hydroxy-2-acetylaminofluorene
RL
16
+++
N-Hydroxy-2-acetylaminophenanthrene
RL
10
+++
N-Hydroxy-4-acetylaminobiphenyl
RL
10
+++
N-Hydroxy-4-acetylamino-trans-stilbene
RL
9
+++
4-Aminobiphenyl
MS
1
+
Benzidine
MS
3
+
Azo compounds
4-Dimethylaminoazobenzene
MS
2
+
Congo red
MS
2
+
Evan's blue
MS
1
+
Nitro compounds
4-Hitroquinoline-l-oxide
MS
8
+
2,6-Dinitrotoluene
MS
3
—
Polycyclic aromatic hydrocarbons
Benzo(a)pyrene
MS
5
+++
RL
2
++
7,12-Dimethylbenz(a)anthracene
MS
8
+++
3-Met hy1cho1anthr ene
MS
13
+++
Benzo(e)pyrene
MS
5
+
Benz(a)anthracene
MS
2
+
Di benz(a,c)anthracene
MS
6
+
Dibenz(a,h)anthracene
MS
3
++
Benzo(g,h,i)perylene
MS
2
++
Chrysene
MS
++
Anthracene
MS
NDd
—
Pyrene
MS/ML
MDd
-
Perylene
MS
ND
-
Benzo(a)fluorene
MS
5
+
Benzo(b)fluorene
MS
5
+
Heterocyclic polycyclic compounds
Dibenzo(c,g)carbazole
MS
7
+++
Dibenzo(a,i)carbazole
MS
6
+
Dibenzo(a,J)acridine
MS
2
++
(continued)
20
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TABLE 3. (continued)
COMPOUND
Alkenylbenzenes
Safrole
Estragole
Nethyleugenol
Hyristicin
Dill apiol
Parsley apiol
Isosafrol
Elemlcin
Anethole
Allylbenzene
Hethylating agents
N,N-Dimethylnitrosamine
1,2-0imethylhydrazlne
N-Methyl-N-nitrosourea
Streptozotocin
Nycotoxins
Aflatoxin Bl.
Sterigmatocystin
DNA adducta
TISSUE No.b Levels
ML 4 +++
ML 4 +++
ML 4 +++
ML 3 +++
ML 3 +++
ML 3 +++
ML 2 ++
ML 2 +++
ML 2 ++
Ml 2 ++
ML 5 Mil
ML 5 ++++
Ml 5 -H-H-
ML 5 Itll
RL 9® ++++
RL 15 +++
^MS, mouse skin; ML, mouse liver; RL, rat liver. Adapted from (180).
These numbers reflect the total number of adducts detected Including
those requiring very prolonged exposures for their detection.
Total adduct levels: +, 1 adduct in > 102 nucleotides; ++, 1 adduct
in 5 x 105. - 101 nucleotides; +++, 1 adduct in 104 - 5 x 10§. nucleotides;
++++, 1 adduct in <104 nucleotides.
dNot detected.
These adducts are oligonucleotides containing covalently bound
carcinogen.
21
-------
can be run per day and as such is a suitable method for screening large
numbers of samples, and the method has been In use for several years. ^
Immunoassays do have a few problems. The methods are not as sensitive as P-
postlabellng and nay not be suitable for low exposure situations where sample
size is limited. In addition, the antibodies must be characterized to
determine what, If any, other adducts cross-react with a particular antibody.
Radiolabeled carcinogens allow for the detection of minute amounts of
carcinogen binding to DNA but are not suitable for human studies. A method
has been developed that has the sensitivity advantage of radiolabeled
compounds, and it does no^2re1uire that the carcinogen be radiolabeled. The
method is referred to as P-postlabellng, and the method is summarized as
follows. Adducted DNA is ^olated from a tissue source and is digested to
form 3'-mononucleotldes. P is incorporated on the 5'-end of the
nucleotides, and the adducts are separated by using multidimensional thin
layer chromatography. This method has been used to screen over 70 compounds
for their DNA adduct forming ability (76, 180, 181; Table 3). The separated
adducts are quan^fled by using autoradiography. Because of the high specific
activity of the P, this method can detect adducts at about one adduct per
10 bases from a 1 ug sample of ONA (181). If the normal nucleotides can be
removed befor^postlabeling, the detection limit can be lowered to about one
adduct per 10 nucleotides (74), to make this one of the most sensitive
methods available. This level of sensitivity may be required when one is
looking for DNA adducts induced by environmental carcinogens in the general
population because of low exposure situations. Another advantage of this
method is that one does not have to know the structure of the adduct in order
to detect or quantltate it. In addition, a characteristic "fingerprint" is
obtained for each adduct-forming compound. It is unclear how useful this
"fingerprint" will be when adducts formed from several compounds are mixed
together. Standards will be required to positively Identify these presumably
complex mixtures in order to establish^ connection between adduct formation
and exposure to a particular method. P is a very strong beta emitter
(approx. 1.7 MeV) and has a mean free path in air of about 20 feet. Stringent
precautions must be taken in order to protect technicians a^g other laboratory
personnel from radiation exposure. Also, the half life of P is abou^l^
days so analyses must be carefully coordinated so as not to waste the P.
22
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LITERATURE REVIEW OF CARCINOGEN DNA ADDUCT STRUCTURE,
PERSISTENCE, AND DOSE/RESPONSE CHARACTERISTICS
This section reviews ONA adduct research that has been published in the
last 5 years. The review will be divided by chemical class and will be
further subdivided by chemical. In addition, the information is summarized in
tabular form in the appendix.
ARYLAMINES
An excellent review of the literature pertaining to the study of
arylamlne-DNA adducts has recently been published (203) and will be summarized
here. Exposure to aromatic amines was first associated with human bladder
cancer in dyestuff industrial workers, and much research has been conducted in
the area of arylamine-induced genotoxicity.
1-NAPTHYLAMINE
Although 1-napthylamine (1-NA) is not carcinogenic, it can be chemically
altered i_n vitro to form N-hydroxy-l-NA. N-hydroxy-l-NA is strongly
carcinogenic at the local site of injection. When N-hydroxy-l-NA was^reacted
in vitro with DNA, two adgucts were characterized: N-(deoxyguanosin-0 -yl)-l-
NA and 2-(deoxyguanosin-0 -yl)-l-N£. When N-hydroxy-l-NA was injected into
rats (16 umol), g-(deoxyguanosin-0 -yl)-l-NA was the major adduct detected
(22.6 adducts/10 nucleotides). The adduct was persistent; adduct levels
dropped only 30 percent from one day after administration to seven days after
administration (60). The reason 1-NA is not carcinogenic is probably because
it is not metabolized to N-hydroxy-l-NA by the cytochrome P-450 monooxygenase
system JUi vivo.
2-NAPTHYLAMINE
2-Napthylamine is a commonly used intermediate in the dyestuff industry.
In contrast to its isomer, 1-NA, 2-napthylamlme (2-NA) is carcinogenic in
rats, dogs, and humans. Like 1-NA, its N-oxidized derivative N-hydroxy-2-NA
formed derivatives with DNA when reacted in vitro (105). Three adducts were
characterized; an Imidazole ring-opened derivative of N-(deoxyguanosin-8-yl)-
2-NA, l-(deoxyguanosln-N -yl)-2-NA, and l-(deoxyadenosin-N -yl)-2-NA. After
administration of 2-NA to dogs (60 umol/kg), the three characterized DNA
adducts wege detected in both the liver (non-target organ; between 0 and 2
adducts/10 bases) and the bladder urothelium [i.eg, the cells lining the
bladder (target organ; between 0 and 10 adducts/10 bases)] (33, 106). The
levels of the adducts were four times higher in the target versus the non- ^
target organ. Seven days later, binding was measured in both organs. The N -
dgoxyguanosine adduct persisted in both the liver and urothelium whereas the
C -deoxyguanosine adguct only persisted in the urothelium (33, 106). The
persistence of the C -deoxyguanosine adduct in the bladder is therefore
associated with the induction of bladder cancer and it may be that this adduct
triggers a mutational event that is important in the initiation of bladder
cancer.
23
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4-ACETYLAMINOBIPHENYL (AABP) and 4-AMINOBIPHENYL (ABP)
4-Acetylaminobiphenyl (AABP) is a research chemical that is a mammary
gland carcinogen in the rat. The presumed active metabolite of AABP is N-
hydroxy-AABP. Attempts were made to characterize the adducts formed in liver
and in mammary gland DNA (123b). Although acetylated and nonacetylated
adducts were found, only the acetylated adduces were characterized. The
adducts were identified as 3-(deoxyguanosin-N -yl)-AABP and N-(deoxyguanosin-
8-yl)-AABP. In another s^dy, the persistence of these adducts in rat liver
was monitored by using a P postlabeling technique (Dose-40 mg/kg). The
adducts detected.were N-(deoxyguanosin-8-yl)-AABP (1.5 fmol adduct/ug DNA), 3-
(deoxyguanosin-N -yl)-AABP (2.4 fmol adduct/ug DNA), N-(deoxyguanosin-8-yl)-4-
aminobiphenyl (ABP) (13.2 fmol adduct/ug DNA), and another unidentified
nonacetylated adduct (75). The levels of these adducts were decreased 80,
62, and 77 percent 24 hours after an acute administration of N-hy^roxy-
acetylaminobiphenyl. However, after 29 days, 38 percent of the N adduct
still remained. Therefore, it appears that at least one of the adducts of
AABP is very persistent.
4-Aminobiphenyl (ABP) is another research chemical that is a potent
bladder carcinogen in dogs and that forms DNA adducts. When N-hydroxy-ABP,
which is the active form of ABP, was reacted with DNA in. vitro, three adducts
were formed; N-(deoxyguanosin-8-yl)-ABP, N-(deoxyguanosin-N -yl)-ABP, and N-
(deoxyadenosin-8-yl)-ABP (33). When dogs were treated with ABP, (60 umol/kg),
the same three adducts were detected in gog bladder and liver DNA (adduct
levels were between 0 and 800 adducts/10 nucleotides) (33). It is
interesting to note that the levels of binding in both organs were high and
essentially the same, but only the bladder is susceptible to this carcinogen.
The persistence of total adduct levels was measured over 7 days. Adduct
levels remained constant over the 7-day period. This is another instance
demonstrating that DNA adduct levels alone are not an indicator of cancer
risk, at least over the 7-day period measured.
4-ACETYLAMIN0-4'-FLU0R0BIPHENYL
If one adds a 4'-fluoro group to AABP, the compound becomes a renal,
hepatic, and mammary gland carcinogen in rats. 4-Acetylamino-4'-fluoro-
biphenyl is used as a cancer research chemical. As one would predict by using
the previous compounds as an example, N-hydroxy-4-acetylamino-4'-
fluorobiphenyl (AAFBP) is the ultimate carcinogen (123a). In the rat both
acetylated and nonacetylated adducts were detected, but only the acetylated
adducts were characterized. The two acetylated adducts were identified as N-
(deoxyguanosin-8-yl)-AAFBP. The persistence of the metabolites varied greatly
(123a). The nonacetylated adducts were removed with a half-life of about 10
days; N-(deoxyguangsin-8-yl)-AAFBP was removed with a half-life of two days.
3-(deoxyguanosin-N -yl)-AAFB appeared to be a persistent lesion at least over
the time frame measured.
3,2'-DIEMTHYL-4-AMIN0BIPHENYL
3,2'-Dimethyl-4-aminobiphenyl (DMABP) is an arylamine research chemical
that is a colon carcinogen in rats. The active metabolite Is N-hydroxy-DMABP.
With K-acetyl-N-hydroxy-DMABP (dose-0.5 mmol/kg),_two adducts were found: N-
(deoxyguanosin-8-yl)-DMABP and 5-(deoxyguanosin-N -yl)-DMABP (229). The
adduct levels in the liver were about twice as high as those seen in the
24
-------
Intestine (60 and 20 pmol adduct/mg ONA versus 40 and 10 pmol adduct/mg DNA,
respectively). However, the basal rate of cell division was at least 20~times<
higher in the Intestine than in the liver, so the adducts in the Intestinal
lining cells are much more likely to cause an error in replication. Because
DMABP is a colon carcinogen but is not a liver carcinogen, the results
indicate that the rate of cell division may be an important consideration is
estimating carcinogenic risk.
2-ACETYLAMIN0FLU0RENE
2-Acetylaminofluorene (AAF) is a biochemical research chemical that is a
carcinogen in a number of species and in a number of organs including the
liver, mammary gland, Intestine, and bladder. N-Hydroxy-AAF appears to be the
ultimate carcinogen, and after a single administration of AAF or N-hydroxy-AAF
to the rat (dose-15 mg/kg), the following adducta were detected in liver; N-
(deoxyguanosin-8-yl)-AAF and 3-(deoxy-guanosin-N -yl)-AAF (123, 124). The
first adduct had a half-life of 7 days whereas the second adduct was a
persistent lesion in hepatic DMA. Later studies (141, 223) indicated the
presence of one major nonacetylated adduct, N-(deoxyguanosin-8-yl)-2-
aminofluorene (AF). This adduct was relatively persistent in rat liver ONA
(31, 76, 141, 175). 2-AAF is one of the few compounds for which long term
dosing studies have been performed. Sprague-Dawley rats were administered 2-
AAF at biweekly intervals for up to 56 days (10 mg/kg; 31), and adduct levels
were measured on each day of dosing and 14 days after each dosing. Male rats
showed all three AAF adducts in hepatic DMA. M-(Deoxyguanosin-8-yl)-AAF was
detected in male rats onl£ and only on the day of dosing (1-1.8 pmol AAF/mg
DNA). 3—(Deoxyguanosin-N -yl)-AAF was detected on days 1 and 14 and increased
with additional administration of 2-AAF (0.2-3 pmol AAF bound/mg DNA). N-
(Deoxyguanosin-8-yl)-AF was the major adduct observed in males (10—20 pmol AAF
bound/mg DMA), and the levels remained approximately constant over 56 days.
The female rats showed only the nonacetylated adduct, and it too was
persistent (10-60 pmol AAF/mg DNA, from day 1 to day 56). Total adduct levels
in the female were higher than in the male; however, female rats are resistant
to 2-AAF-lnduced carcinogenesis. This again brings up the point that total
DNA adduct levels alone are not directly correlated to cancer Incidence. In
another study (175, 176), rats were given either 0.02 or 0.04 percent AAF.
Adduct levels increased with time until equilibrium (about 300 pmol adduct/mg
DNA) was reached; equilibrium was reached in about three weeks. Both
acetylated and nonacetylated adducts were detected Initially although when
equilibrium was reached, only nonacetylated adducts were present. Adduct
formation was also detected in the kidney and mammary gland. In single
administration and continuous dosing studies, only M-(deoxyguanosin-8-yl)-AF
was detected in these tissues (6, 31, 175).
2-AAF also Induces bladder and liver tumors in dogs (60 umol/kg). N- 0
(Deoxyguanosln-8-yD-AF was detected in both tissues (100 and 500 adducts/10
nucleotides, respectively); other minor adducts were detected in hepatic DNA
(33). However, between days 2 and 7 after administration, 80 percent of the
adducts were removed. Mice are susceptible to hepatic tumors when exposed to
2-AAF, and N-(deoxyguanosin-8-yl)-AF was the major adduct found in heptatlc
DNA (127).
A related compound, 7-fluoro-2-acetamldofluorene, has been studied in
rats, and one adduct has been characterized [8-(N-fluorenylacetamldoguanlne
adduct] (189). However, this compound is a laboratory-created test compound
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with no real environmental significance.
BENZIDINE
Benzidine (BZ) Is a compound that Is the building block of an entire
family of dyes known as benzidine dyes. Benzidine is a urinary bladder
carcinogen in humans and dogs and a hepatocarcinogen in rats, mice, and
hamsters. It is believed that benzidine is converted enzymatlcally to the
carcinogenic molecules N-hydroxy-N'-acetyl-BZ and N,N'-dlacetyl benzidine.
The major l_n vivo adduct found in the rat (dose-Ill uinol/kg) was N-
(deoxyguanosin-8-yl)-N'-acetyl-BZ (70 pmol adduct/mg DNA) with lesser amounts
of N-(deoxyguanosln-8-yl)-N,N'-diacetyl-BZ present (5 pmol/mg DNA; 115, 138,
139). The persistence of the major adduct in the rat has been measured in two
studies. One study showed a 60 percent decrease in binding from day 1 to day
7 after administration of N-acetyl-BZ (initial adduct level in rat liver-90
pmol BZ/mg DNA; dosed for one week in drinking water-80 ppm; 138). The second
study found a 40 percent decrease in adduct binding between days 1 and 2 after
administration with no further decrease in binding thereafter for at least 4
weeks (137a). Similar results are seen in the mouse. A single adduct was
found in the mouse; N-(deoxyguanosin-8-yl)-N'-acetyl-BZ. Adduct levels were
highest immediately after cessation of dosing, and although adduct levels
dropped 50 percent after one day, adduct levels remained constant for at least
another week (138). In hamsters, the above adduct was found in hepatic DNA,
and its persistence was similar to that seen in the rat (115). After dosing
at 111 umol/kg, the level of binding of the above adduct after one day in
liver DNA was 33 pmol/mg DNA. After 7 days, the binding of BZ dropped about
60 percent. Benzidine forms bladder DNA adducts in dogs, but the adducts have
not as yet been characterized. Most dyes that form covalent DNA adducts are
metabolized to benzidine, and, therefore, benzidine adducts are detected.
However, this is not always the case. Although the dye Direct Blue 6 does
yield N-(deoxyguanosin-8-yl)-N'-acetyl benzidine, another larger adduct has
been detected and characterized (116). The adduct was characterized as
di sodiurn 8-amino-2-[4-(N-deoxyguanosin-8-y1)-aminobipheny1-4'-yl] azo-1-
hydroxy-naphthalene-3,6-disulfonate. Therefore, if one is going to study
azodye-DNA adducts, one might also want to measure the azo dye-DNA adducts
instead of benzldine-DNA adducts as they would be indicative of the original
dye from which the adduct was derived.
N,N'-DIMETHYL-4-AMIN0AZ0BENZENE, N-METHYL-4-AMIN0AZ0BENZENE, AND 4-
AMINOAZOBENZENE
N,N'-Dimethyl-4-amlnoazobenzene (DAB) is a dye that was one of the first
arylamlnes studied in terms of adduct formation. DAB Is more commonly known
as Butter Yellow. Metabolic activation occurs by demethylation to form N-
methyl-4-amlnoazobenzene (MAB). MAB is then N-oxidlzed to form N-hydroxy-MAB.
The ultimate carcinogen is believed to be N-sulfonyloxy-HAB. tlAB has been
used In most studies because it is more carcinogenic than 4-amlnoazobenzene
(AB). In the rat the major hepatic adduct found was N-(deoxyguanosln-8-yl)-
MAB (135). However, this adduct Is not persistent; 100 percent of the adduct
is removed afte^? days (30, 32, 135). The minor adduct found, 3-
(deoxyguanosln-N -yl)-MAB, was persistent; its level remained constant for at
least 2 weeks (30, 32). If multlple^doses of MAB were administered, a third
adduct was found: 3-(deoxyadenosin-N -yl)-MAB (216). Rats were administered
MAB at a level of 0.2 nmol/kg on days 1,3,5 and 8. The adduct levels
increased from 0 to 2 adducts/106 nucleotides over the 8 days. In a long-term
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(5 week) feeding study (217), all three adducts were found to increase with
time. However, when dosing was suspended, only one of the three adducts, 3-
(deoxyguanosin-8-yl)-HAB, was persistent. Similar results were found in the
mouse; 3-(deoxyguanosin-8-yl)-MAB was a much more persistent lesion in hepatic
DNA than N-(deoxyguanosln-8-yl)-MAB (213).
AB is carcinogenic in very young mice, and one adduct has been
characterized in mouse hepatic DNA when the mice were administered 0.3 umol
AB/gi N-(deoxyguanosln-8-yl)-AB at a level of 20 pmol adduct/mg DNA (54).
Vhen the same type of mice were administered DAB (0.3 umol/g), the above
adduct was found (5.5 pmol/mg DNA) along with N-(deoxyguanosin-8-yl)-MAB (2.8
pmol/mg DNA) and 3-(deoxyguanosin-8-yl)-MAB (1.5 pmol/mg DNA).
2-ACETYLAMIN0PHENANTHRENE
Although 2-acetylaminophenanthre^ (AAP) is not carcinogenic, it binds to
DNA both In vitro and in. vivo. In a P postlabeling study, it was found that
the hepatic DNA adduct levels of AAP increased with time up to 24 h to 61
pmol/mg DNA and only decreased 50 percent after 29 days (75) after a single
intraperitoneal injection of N-hydroxy-AAP (40 mg/kg). Many adducts were
detected, but only one was characterized; N-(deoxyguanosin-8-yl)-
aminophenanthrene. The same study showed that DNA adduct levels induced by 2-
AAF, a potent carcinogen, were removed by about 95 percent 29 days after
injection. The results suggest that even adduct persistence is not
necessarily followed by tumor formation.
trans-ACETYLAMINOSTILBENE AND trans-4-DIMETHYLAMINQSTILBENE
trans-Acetylaminostilbene (AAS) and trans-4-dimethylaminostilbene (DAS)
are research chemicals that are used as model arylamines. AAS and DAS are
potent carcinogens in rats, particularly in the sebaceous glands of rats. A
long-term (6-weeks) feeding study with rats showed that AAS bound to DNA,
protein, and RNA in several organs in the rat, but there was no correlation
between binding levels of the adducts and organ sensitivity to AAS (93). Vhen
organ exposure, as determined by protein adduct formation, was measured, there
again was no correlation between exposure and organ sensitivity. It would
have been Interesting to measure DNA repair rates to see if any correlation
existed between DNA repair and organ sensitivity.
trans-4-Dlmethvlaminoatllbene (DAS) was employed to study the dose-
dependence of adduct formation. The compound was administered to rats in
doses ranging from 5 x 10 to 1 x 10 mol/kg. Binding of DAS to protein,
DNA and ribosomal RNA was linear, and binding levels ranged from 0.5 to about
100,000 fmol/mg macromolecule (155). This report is important in that it
provides experimental evidence that shows a carcinogen dose-response
relationship can be linear over several orders of magnitude. It suggests that
It should be possible to obtain usable dose-response relationships for
carcinogens at the low levels to which people might be exposed.
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POLYCYCLIC AROMATIC HYDROCARBONS
Polycyclic aromatic hydrocarbons (PAH) are widespread environmental
pollutants that are produced primarily by industrial and transportational
sources and can be found in materials such as soot, coal, tobacco smoke, and
petroleum products. Because these compounds are carcinogenic in laboratory
animals and because human exposure to these chemicals is widespread, much
research has taken place in this area to determine if PAH's are carcinogenic
in man. Epidemiologic studies have shown that industrial exposure to PAH's is
associated with higher incidences of lung, skin, and bladder cancers (90, 112-
114, 200). Cigarette smoking (16) and living in areas with high pollution
(16, 89, 103, 172) are also associated with increased lung cancer incidence.
All of the above conditions Involve exposure to PAH's. The following is a
summary of the research to date that characterizes the formation and
persistence of PAH's. For a more detailed summary, an excellent review has
been written by Stowers and Anderson (203).
BENZ0(a)PYRENE
Benzo(a)pyrene (BaP) is the most extensively studied of the PAH's and is
one of the most widespread in the environment. BaP is so widespread primarily
because it is almost always associated with combustion. In our post-
industrial revolutionary age, BaP is ubiquitous. It was one of the first
carcinogens studied because it can be measured by using its UV absorption
properties. BaP is a PAH that must be metabolized before it can form covalent
adducts with DNA. After two epoxidation steps, the two major BaP metabolites
that bind to DNA are (+)-7B,8a-dihydroxy-a,10a-epoxy-7,8,9,10-
tetrahydrobenzo(a)pyrene (BPDEI) and (—)—7B,8a-dihydroxy-9B,lOB-epoxy-
7,8,9,10-tetrahydro-benzo(a)pyrene (BPDEII). These types of metabolites are
known as diol epoxides, and most other PAH metabolites are of the same type.
Studies have also been conducted by using human colon tissue to measure
metabolism of BaP in humans. The two major metabolites found were those
listed above (13).
BPDEI and BPDEII mainly bind to the 2-amino group of guanine residues,
but they can also bind to the N-7 of guanine (159), adenine (108, 109, 140,
204), cytosine (204), and to phosphate residues (69, 121). BPDEI and BPDEII
bind to lung, liver, colon, kidney, muscle, brain, and forestomach of the
A/HeJ mouse and to lung, liver, colon, muscle, brain, and blood of the New
Zealand White Rabbit (203). Other studies show adduct formation in lung,
liver and kidney, colon, and intestine of mice (61). BaP also binds to DNA in
the skin of several mouse strains (11, 18, 45, 52, 73, 122, 163, 168). The
major adduct found in all cases was BPDEI bound to the N-2 position of guanine
residues. Minor adducts are also seen bound to adenine residues. Once again,
similar adduct formation is found in both susceptible and resistant organs.
Adduct formation In the rat has also been measured; however, all of the
adducts have not been characterized. One adduct has been identified in the
rat (BPDEI-guanlne). In rat lung, liver, and skin, it was a minor adduct (19,
39, 102) whereas in rat hepatocytes, it was the major adduct (10). Adducts
have also been detected in humans (63,165)
Several dose-response studies have been conducted. In several mouse
strains and in several organs, BaP levels in various organs have varied
linearly (0.01-300 ug BaP/mouse, 2-11 umol/kg in the mouse; 3, 163) and non-
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linearly (11-135 umol/kg in the mouse, slgmoldal dose./respones;3). In both
studies, the dose response curve was linear or approached linearity at the
lower doses. If this linearity response at low doses is a general phenomenon)
for most carcinogens, it may be relatively easy to establish a dose-response
relationship in a human pilot study.
In the rat, an increase of an intravenous administration of BaP from 1
umol to 10 umol increased adduct levels in rat lung approximately 5 fold and
in rat liver about 3 fold (39). Although dose response linearity may not
exist at high dose levels, it appears that the association of low levels of
adducts with a given dose should be possible. One of the most interesting
studies measured DNA adduct formation in cultured human, monkey, dog, hamster,
and rat tissue [bladder and tracheobronchial tissues] (51). The patterns of
metabolism were similar in all species; however, the level of adducts were
widely variable. ONA adducts in human bladder were 30 times higher than in
the rat. In the trachea-bronchus, the difference in adduct binding was about
10 fold between man and the rat.
BaP binding to proteins, both cellular (3) and circulating [i.e.,
hemoglobin (195)], has been measured. The dose-response curve was linear in
both cases, and the increase in BaP-protein adducts correlated with the
increase in BaP-DNA adducts. Therefore, measurement of BaP-protein adducts
would be a good method for monitoring exposure to BaP in the general
population. Studies have been run on human populations. One study measured
BaP adduct formation in exposed and unexposed coke oven workers. Although the
exposed group showed higher DNA adduct levels than the unexposed group,
individual variation was great. Some exposed workers showed no measurable
adduct formation (C.C. Harris, personal communication). This suggests that an
estimate of individual DNA repair rates, enzyme levels, etc., may be necessary
in order to interpret the ONA adduct level information.
The persistence of BaP-ONA adducts varies from species to species and
from organ to organ; however, there is no correlation between adduct levels
and the susceptlblity of a particular strain or organ to the carcinogenic
effects of BaP (61, 125, 161, 168, 95). The following are examples of some of
the studies that measured persistence of adducts. Pelling et al. (162a)
measured the persistence of the (+) (active) and (-) (inactive) forms in mouse
skin ONA. The adduct persistence for both compounds was about 2-3 days; the
carcinogenic potential of these compounds differs by about 60 fold.
Therefore, carcinogenic potential does not appear to be related to the
persistence of BaP adducts in this tissue and mouse strain. Another study
compared the persistence of several BaP adducts in mouse and rat skin
epidermis (5). The main adduct, BPDEI-dG, had a half-life of less than a week
in both species. However, 6 percent of the adduct remained in the mouse skin
ONA after 3 weeks whereas the same adduct was completely removed after the
same period of time in the rat. This shows that the simple measurement of a
half-life may not give the entire story of persistence. Persistence very
often tends to be biphaslc with an initial rapid decrease in adducts which is
followed by a slower adduct removal rate. Kulkarni and Anderson (125)
measured the persistence of BP0EI-0NA adduct formation in the lung and liver
of A/HeJ and C57BL/6J mouse strains. The half-life of the adducts in the
A/HeJ mice were 20 and 14 days in the lung and liver whereas the half-life of
the adducts in the C57BL/6J were 16 and 3 days, respectively.
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The related compound 10-azabenzo(a)pyrene-4,5-oxide is a mutagen that can
form DNA adducts (157) but that has no environmental significance. Another
model compound that has been used to study PAH-induced adduct formation is 9-
anthryloxirane. An in vitro study that reacted this compound with calf thymus
DNA showed that the major adduct formed was through the N-3 position of
adenine (237). Physicochemical studies have been conducted with the compound
1-oxiranylpyrene (117). This is a model compound that mimics the dlol-epoxlde
of BaP but that does not contain any hydroxyl groups. This compound, although
useful in kinetics experiments and other related experiments, has no
environmental significance.
3-METHYLCHOLANTHRENE
3-Methylcholanthrene (3-MC) is a PAH that is commonly used in
toxicological and pharmacological research as an inducer of the cytochrome P-
450 monooxygenase system. It is also a carcinogen and is believed to be
activated through a dlol-epoxlde in a manner similar to that seen with BaP
(118, 119, 215). 3-MC has been shown to form DNA adducts In cultured human
bronchus, colon, esophagus, pancreatic duct, and bladder (77). One study on
the 3-MC-DNA adduct persistence in the mouse has been conducted. The
persistence of adducts in four different mouse strains was measured. Adduct
levels were measured at 4 hours, 7 days, and 28 days. In the lung, a
susceptible organ designated as the A/J, C3H/HeJ, DBA/2J, and C57BL/6J mouse
strains all showed persistence of DNA adducts from 19 to 62 percent after 28
days. In the liver, which is a resistant organ, all 3-MC adducts were removed
after 28 days. This experiment suggests that there is a correlation between
adduct persistence and the susceptibility to cancer. This is the exception
rather than the rule concerning PAH adduct levels.
7,12-DIMETHYLBENZANTHRACENE
7,12-Dimethylbenzanthracene (DMBA) Is one at the most powerful synthetic
PAH carcinogens known. The binding and persistence of DMBA has been studied
in mouse skin, rat liver, and cultured human tissue. DMBA forms DNA adducts
in human bronchus, colon, esophagus, and pancreatic duct cultured cells (203);
however, the adducts were not characterized nor quantified. Daniel and Joyce
(50) injected Sprague-Dawley (susceptible) and Long-Evans (resistant) strains
with DMBA (20 umol/rat). Five adducts were detected, but only two were
partially characterized. The adducts were identified as DNA adducts with
1,2,3,4-tetrahydro-7,12-dimethylbenz(a)anthracene-3,4-diol-l,2-epoxide and
with the diol-epoxide metabolite, 7-hydroxymethyl-12-ethylbenz(a)anthracene.
Maximum levels of DMBA adducts were found in mammary gland DNA of the
resistant and susceptible strain after 24 hr (20 and 18 umol DMBA/mol DNA,
respectively). Vhen persistence was measured up to 12 days after injection,
the susceptible strain did not repair the adducts whereas limited adduct
repair occurred in the resistant strain. Vatabe et al. (226, 227) reported
that the sulfate esters of hydroxymethyl-methyl-benz[a]anthracenes were
mutagenic in the Ames assay. Dlpple et al. (59) measured binding of DMBA to
mouse skin DNA in two strains of mice, NIH Swiss and C57BL, after dermal
application of 0.01 and 0.1 umol DMBA. Partial characterization of three
adducts showed the formation of an anti-deoxyguanoslne adduct and both a-svn
and antl-deoxvadenoslne adduct. The distribution of adducts was similar in
both strains, with maximum adduct levels reached at 24 hours and with total
adduct levels of 4.6 and 5.45 umol adduct/mol DNA (0.1 umol dermal
application). At 48 hours, some decrease in adduct levels occurred. Total
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adduct levels dropped from 9 to 26 percent, the different levels were
dependent on dose and strain. It should be noted that a ten-fold increase in
the dermally-applied dose led to a 3- to 4-fold increase in the total ONA
adduct levels.
15,16-DIHYDRO-ll-METHYLCYCLOPENTA[a]PHENANTHRENE-7-ONE
15,16-dihydro-ll-methylcyclopenta[a]phenanthrene-7-one (11-methyl ketone)
is a PAH that is carcinogenic in mouse skin and lung, but not in mouse liver.
Several studies on the metabolism of 11-methyl ketone indicate that the
major carcinogenic metabolite formed is a 3,4-dihydrodiol-l,Z-epoxide (1, 47).
The major ONA adduct appears to Involve covalent bonding between the N£ of
guanine and the C-l carbon of 11-methyl ketone (231). One study has been
conducted that determined the persistence of the ONA adducts produced by 11-
methyl ketone in mouse skin, lung, and liver (2). Mice were injected with 3
mg of 11-methyl ketone, and the time couse of adduct removal was monitored for
two weeks. Maximal adduct formation occurred about 2 days after injection.
Total binding in the skin, liver, and lung was 283, 345, and 641 pmol
adduct/nunol ONA. The half-life of the adducts in both skin and lung was about
6.5 days whereas the half-life of the adducts in the liver was about 2.5 days.
When the DNA turnover rates were taken into account, it appeared that the lung
and skin lesions were much more persistent than the lesions in the liver. It
should be noted that maximal ONA adduct levels in the liver were twice as high
as those seen in either the skin or lung; this suggests that rates of cell
division are much more important than initial ONA adduct levels.
7-BR0M0BENZANTHRACENE
One study has been located on this compound that measured the dose-
response and persistence of 7-bromobenzanthracene (7-BMBA)-DNA adducts in
mouse liver DNA, ONA associated proteins, and serum proteins (24). Mice were
given an intravenous injection of 7.7 nmol 7-BMBA per gram body weight, and
the time course of adduct formation was measured. Adduct formation reached a
maximum in all targets measured in 0.5 to 3 hours, and adduct levels reached
about 40 adducts/lOl nucleotides in DNA. The dose-response curves for 7-BMBA
was nonlinear for both proteins and DNA (24). Most of the 7-BMBA was rapidly
removed from the mouse liver DNA within 1 day; however, a low level of 7-BMBA
persisted for at least 1 month. 7-BMBA levels in albumin decreased rapidly
with the loss of adduct corresponding to the normal turnover of the serum
protein. Adduct levels decreased in the histone proteins, but it is not clear
if the decrease represents protein turnover or adduct removal from the
protein.
DIBENZ(a,h)ANTHRACENE
Dlbenz(a,h)anthracene is a product resulting from incomplete combustion of
organic materials. The only information located on DNA adducts formed by
dlbenz(a,h)anthracene is that adducts are Indeed formed when cultured human
bronchus, colon, and esophagus cells are Incubated in the presence of this
compound (77).
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DIBENZO(a,e)FLUORANTHENE
Dibenzo(a,e)fluoranthene (DBF) Is a PAH that binds to DNA. In an l_n vitro
study, two dlol-epoxlde forms of DBF were reacted with calf thymus DNA (166).
Acid hydrolysis and analysis of the nucleotides revealed that the active forma
of DgF were reacted specifically with the exocycllc nitrogen of guanine (i.e.,
an N guanine adduct). When mouse embryo fibroblasts were reacted with DBF,
at least 6 adducts were formed (167). The persistence of 5 of the adducts was
monitored for up to 48 hours. Some of the adducts were removed with an
approximate half-life of 48 hours; however, others did not appear to be
removed over the 48 hour period (167).
5-METHYLCHRYSENE
5-Hethylchrysene (5-MC) is an environmental carcinogen that is found in
tobacco smoke (158). This compound has two 'bay regions,' and, as one would
expect, two active metabolites of 5-MC have been identified; both are dlol-
epoxldes located at each bay region (144). Both compounds reacted with DNA In
vitro, and the linkage to DNA was through the exocycllc nitrogen of guanine
residues (144). These same adducts were detected In mouse skin DNA after
dermal application of 5-MC (142). The two aforementioned adducts were
detected in a ratio of 2.7:1 (3,4-epoxy versus 9,10-epoxy). Time course and
persistence studies were conducted, and it was determined that the difference
in adduct levels was attributable to differing levels of reactivity of the two
dlol-epoxides (143). The two adducts described were the major adducts
detected, but other uncharacterlzed adducts were also present. 5-MC can be
metabolized to 5-hydroxymethylchrysene (158). Okuda et al. (158) have
reported that 5-hydroxymethylchrysene, when converted to a hydroxymethyl
sulfate ester, is mutagenic in Salmonella typhlmurium. This Indicates that
there are other metabolites in addition to the dlol-epoxides that can form DNA
adducts.
1-NITROPYRENE
1-Nitropyrene (1-NP) is a PAH that is found in diesel exhaust. Metabolic
studies with 1-NP in Salmonella typhlmurium and Chinese hamster ovary cells
Indicated that 1-NP undergoes nltroreductlon to 1-amlnopyrene (4, 96). The
Implication Is that the reactive Intermediate is N-hyroxy-l-amlnopyrene.
Several adducts have been detected In Salmonella, in Chinese hamster ovary
cells, and in rabbit tracheal cells (87, 96, 98), and most have not been
identified (28); but one adduct has been characterized in all of the tissues:
N-(deoxyguanosln-8-yl)-l-amlnopyrene. 1-NP is a mutagen, and a linear
correlation was shown between the level of DNA adducts and the level of
Salmonella revertants (96).
1,8-DINITROPYRENE
Another component of diesel exhaust that has been studied Is 1,8-dinltro-
pyrene (1-DNP). When Salmonella typhlmurium was cultured in the presence of
1-DNP, the major adduct formed was N-(deoxyguanosln-8-yl)-l-amlno-8-nitro-
pyrene (87). This Implies that the reactive intermediate Is l-hydroxyamino-8-
nitropyrene.
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4-NITROQUINOLINE-l-OXIDE
4-Nitroquinoline-l-oxide (4-NQO) Is a carcinogen that must be activated
before DNA adduction will occur. Two compounds have been used as model
ultimate carcinogens of 4-NQO: the 0,0'-diacetyl and the 0-acetyl derivatives
of 4-hydroxyaminoqulnoline 1-oxide (4-HAQO). These compounds, when reacted
with ONA in vitro, formed five detectable adducts (three with guanoslne and
two with adenosine). One adduct was characterized; it was identified as N-
(deoxyguanosln-8-yl)-4-aminoquinoline 1-oxide (21). Uhen the relative rates
of reactivity of the two model ultimate carcinogens were compared, the
monoacetyl derivative was 2- to 3-fold more reactive with DNA (21). The
reactivity of this more active derivative was studied using both native and
denatured DNA. The levels of the one characterized adduct was 3.5 times
higher in the denatured DNA (67). One would expect this, as active sites
would be more accessible to attack in the denatured state. One problem with
studies on this compound, as well as other compounds, is the fact that many
adducts are not characterized. The one characterized adduct accounts for only
30 percent of the total number of adducts present after an In vitro incubation
of the monoacetylated derivative of 4-NQO with denatured DNA (68). In a
recent study, the levels of DNA adduct formation and the persistence of the
adducts were measured in rat pancreas under conditions of high tumorigenicity
(partially pancreatectomized) and low tumorigenicity (non-operated) (56). No
difference was detected In adduct levels or persistence in the high and low
tumorigenicity states. In other words, if the basal rate of cell division is
increased, a given level of DNA adducts may be more likely to Induce tumor
formation. Another study measured unscheduled DNA synthesis (UDS), a measure
of DNA damage, In mouse lung and liver DNA (126). 4-NQO is a carcinogen in
mouse lung but not In liver. Uhen UDS was measured in both tissues, DNA
repair was measured in the lung but was not measured In the liver; this is a
finding which is In apparent contradiction with the carcinogenic potential of
4-NQO. DNA adducts can also cause cell death as well as initiate tumor
formation. Uhen DNA was adducted in vitro with 4-HAQO, Ej. coll DNA
polymerases could not replicate the entire length of the DNA; that is, arrest
of DNA elongation occurred (238).
NATURAL PRODUCTS
There are many natural products, of a diverse nature, that are
carcinogens. Such compounds as fungal and bacterial toxins and products
produced by various plants are known to be carcinogenic. The literature on
these compounds will be summarized in this section unless the compound is a
simple alkylating agent, in which case it will be discussed in the section on
alkylating agents.
AFLATOXIN Bj
Aflatoxln Bj (AFB) is a toxin produced by the mold Aspergillus flavus that
is highly toxic, mutagenic, teratogenic, and carcinogenic in a number of
species (202). Aflatoxlns have been reported to occur naturally in peanuts,
peanut meal, cottonseed meal, corn, dried chili peppers, and other foodstuffs.
Hepatic tumors are Induced In several species after dietary Intake of very low
levels (parts per billion) of AFB. The FDA allows up to 15 ppb of AFB in
foods because the incidence of liver cancer in the U.S. is so low. However,
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in countries where foods contain high levels of AFB, primary liver cancer Is a
relatively common occurrence.
AFB has a complicated metabolic pathway, but it is believed that the two
metabolites that are responsible for AFB-DNA adduct formation are the 8,9- and
2,3- epoxide forms of AFB (8, 49, 163, 202). N -AFB adducts have been
detected In several species, as has the imidazole ring-opened fonp of the
above adduct (8, 43, 48, 49, 163, 202). The persistence of the N -AFB adduct
is not great; it is less than 2 days, but the ring-opened adduct is very
persistent; no loss of the adduct was measured over a 72 hour period after
treatment of mouse embryo fibroblasts with AFB (8). In addition to studies
that monitored the formation of DNA-adducts, the removal of ONA adducts has
also been monitored. When rats were administered AFB, AFB-guanine adducts
could be measured in the urine of the animals (35). The levels of adducts in
the urine were nearly linear when compared with the dose (.125-1.0 mg AFB/kg),
and the levels of DNA adducts in the liver correlated almost exactly with the
levels in the urine.
STERIGMATOCYSTIN
Sterlgmatocystln (ST) is another mold-produced toxin that is carcinogenic
in a number of species (183). In a manner similar to aflatoxln B^, ST is
activated by metabolizing enzymes to a 1,2-epoxide form that can react with
the N of guanine (183). Although the ring-opened guanine structure has not
been rigorously identified l£ vivo, it likely exists as it is chemically more
stable than the non-ring-opened form (32, 183). The persistence of the
adducts in rat liver appears to be triphasic. Half-lives of 12 hours, 7 days,
and 109 days were measured (142). The persistence data may suggest that some
adducts are more persistent than others or that some sites on DNA are more
resistant to repair than others.
T-2 TOXIN
Although T-2 Toxin has been shown to bind to the DNA of cultured human
esophagus cells (77), no reports of T-2 Toxin -DNA adducts have been located
on this compound.
MITOMYCIN C
Mitomycin C (MMC) is an antibiotic agent that is also used as an antitumor
agent. MMC forms both monoadducts and DNA crosslinks. After undergoing
reduction, MMC can form covalent adducts. When activated MMC was Incubated
with DNA ^n vitro, three adducts were detectgd. Adducts were formed among MMC
and the 0 and N atoms of guanine and the N atom of adenine (84).
HYMEN0X0N
Hymenoxon (HYM) is a sesquiterpene lactone that is the primary toxic
component in the plant commonly called bitterweed. An ij\ vitro study was
conducted to determine if HYM would bind to DNA. HYM formed adducts with
guanosine, adenine, and cytosine, with guanosine adduct levels > adenine
adduct levels > cytosine adduct levels (209).
34
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FOOD PRODUCTS
There are many compounds in food that are carcinogens, either in their
native form or after food preparation (i.e., cooking). Two reviews give an
overview of this subject and will not be discussed here (7, 224). The
following is a review of food products that form DNA adducts.
TRYPTOPHAN AND GLUTAMIC ACID PYROLYSIS PRODUCTS
Vhen the amino acids tryptophan and glutamic acid are pyrolyzed during the
cooking process, the products formed are 3-amino-l-methyl-5H-pyrido[4,3-b]-
indole (Trp-P-2) and 2-amino-6-methyldipyrido-[1,2-a:3',2'—d]imidazole,
respectively (81-83, 85). These compounds are converted to the active form
via the cytochrome P-450 monooxygenase system to the N-hydroxy derivatives N-
OH-Trp-P-2 and N-OH-Glu-P-1 and have been shown to bind to DNA. The adducts
have been identified as 3-(C -guanyl)-amino-l-methyl-5H-pyrido[4,3-b]indole
(Gua-Trp-P-2) and 2-(C -guanyl)-amino-6-ethyl-dipyrido[1,2-a:3',2'-d]imidazole
(Gua-Glu-P-1) (82, 85). In addition to DNA adduct formation, these compounds
also cause DNA strand scission through an oxidation-type process (225).
ALKENYLBENZENES
A number of alkenylbenzenes are found naturally in many essential oils
that have been or are being used in food preparations or that are found in
certain spices used in food preparations. The two most commonly studied
compounds in this class are safrole and estragole. These compounds, as well
as others of this compound class, are believed activated through the formation
of a 1'-hydroxy1 group. Further metabolism to expoxide, oxo, or sulfonoxy
groups is believed to produce the ultimate carcinogens (170), and both safrole
and estragole follow this metabolic pathway (169, 170, 234). Covalent adducts
have been measured with safrole and estragole in rat liver DNA, transfer RNA,
a^d cellular protein (169, 170^. The DNA adducts have been identified as two
N -deoxyguanosine adducts, a C -deoxyguanosin^adduct» and an N -deoxy-
guanosine adduct (169, 170, 234). Using the P-postlabeling technique,
Randerath et al. (179) have measured the binding of the following
alkenylbenzenes to rat liver DNA: safrole, estragole, methyleugenol,
allylbenzene, anethole, myristicin, parsley apiol, dill apiol, elemicin, and
isosafrole. The presence of some of the safrole adducts was detected at 140
days after administration of safrole (179), and some of the other
alkenylbenzene adducts persisted for at least 43 days after compound
administration (171).
PSORALENS
Psoralens are compounds of plant and synthetic origin that have been used
extensively in clinical dermatology for treatment of diseases such as eczema
and psoriasis. When the compounds are exposed to light at 360 nm, psoralens
can form monoadducts with DNA, with the major adduct being a cycloadduct with
thymine through the 5 and 6 positions (44). This psoralen-induced DNA damage
inhibits or slows cell division and alleviates the symptoms of these
conditions. Some psoralens can also form DNA crosslinks. Psoralens that have
been studied include trimethylpsoralen, 8-methoxypsoralen, angellcln, and 5-
methylisopsoralen. Further information on psoralens can be obtained in recent
reviews of the subject (44, 101).
35
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MOHOCROTALIHE
Honocroatallne is an alkaloid present In the entire p^ant of Crotalarla
apectabllls. Monocrotallne forms an adduct through the N position of guanine
(185).
CC-1065
CC-1065 is a naturally-occurring antibiotic found in Streptomyceg that
can form DNA adducts. CC-1065 forms an unusual adduct through the N position
of adenine, and it may function through inhibition of DNA synthesis (206).
THERAPEUTIC AGENTS
Many therapeutic agents form covalent adducts with DNA. The large
majority of these agents are anticancer drugs. Host of the anticancer drugs
are alkylating agents and will be discussed elsewhere.
CISPLATIN
One anticancer drug that is not strictly an alkylating agent, in the sense
that it does not add an alkyl group to DNA, is cls-dlammlnedlchloro-
platlnum(II) (clsplatin). Cisplatin destroys cancer cells by forming, among
others, bidentate intrastrand N -deoxyguanosine adducts (177). Cisplatin is a
direct-acting carcinogen; that Is, it requires no metabolic activation.
Cisplatin shows linear dose-response relationships in both man and
experimental systems. Cisplatin adduct levels were measured in white blood
cells of patients that had received 100-800 mg cisplatin/M body surface area
and adduct levels ranged from about 30 to 200 fmol adduct/mg DNA (177). Uhen
cultured Chinese hamster ovary cells were treated with 25 to 200 uM cisplatin,
the level of DNA cross-links ranged from 0.3 to 2.5 interstrand DNA cross-
links/109 daltons. The persistence of cisplatin in experimental animals is
blphasic. The first half-life lies between 6 mln and 1.5 hours whereas the
second half-life was between 16 hours and 45 days. Because the dose received
is known, a study of patients where correlations are made between the adduct
levels and the enzyme, glutathione, etc. levelB may be useful, both in
determining whether the use of DNA adducts to estimate exposure is feasible
and to improve the effectiveness of cisplatin in chemotherapy..
DIETHYLSTILBESTROL
Dlethylstllbestrol (DES) is a synthetic estrogen that is a known
carcinogen in humans and experimental animals. Although it is a carcinogen,
little or no binding of DES to DNA has been determined, and, as a result, it
was classified as a non-genotoxlc carcinogen. Llehr et al. (133) conducted a
long-term feeding study with DES in male Syrian hamsters where DES Is a known
renal carcinogen. Although no adducts were detected initially, three adducts
were detected in the hamster kidney, a target organ, but not in the liver, a
non-target organ. Because the measurement procedure does not give any
information about the adduct structure, definite proof does not exist that a
DES-DNA adduct was formed. The only certainty is that DNA adducts of some
sort were formed.
36
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ANTIPSYCHOTIC DRUGS
Two phenothiazine drugs, chlorpromazine and promethazine, have been shown
to bind In in vitro and In bacterial systems to DNA and protein (55). The
compounds were activated both through formation of a radical cation and
through photoactlvation with 350 nm light.
MISCELLANEOUS
The following compounds are a mixture of Industrial chemicals and other
compounds that do not fit into any of the above classes.
ACRYLONITRILE
Acrylonltrile Is a compound that is produced in large quantity and to
which about 300,000 workers are potentially exposed, particularly in the
polymer Industry. A recent study measured the i_n vitro binding of
acrylonltrile to calf thymus DNA (201). The following addicts were detected
by using mass spectrometry: l-(2-carboxy-ethyl)-adenine, N -(2-carboxyethyl)-
adenlne, 3-(2-carboxy-ethyl)-cytosine, 7-(2-cyanoethyl)-guanosine, 7,9-bis-(2-
cyanoethyl)-guanoslne, imidazole ring-opened 7,9-bis-(2-cyanoethyl)-guanosine,
and 3-(2-cyanoethyl)-thymine.
BENZYL CHLORIDE AND 4-CHL0R0METHYLBIPHENYL
Benzyl chloride is a chemical intermediate used in the manufacture of
perfumes, pharmaceutical products, dyes, synthetic tannins, and artificial
resins. No information was located on the sources of 4-chloromethylblphenyl.
The above compounds were found to damage the DNA of xeroderma plgmentosum-
derived fibroblasts, but no information was located on the adducts formed
(147).
STYRENE
Styrene is one of the most Important monomers used in the plastics and
synthetic rubber Industry, and, as such, a fairly large number of workers are
potentially exposed. Styrene requires metabolic activation to styren^ 7,8-
oxlde before DNA adduction can occur. The major adducts formed are N -guanine
adducts through the epoxide moiety (41). When rats were administered styrene
or styrene 7,8-oxlde, adducts were detected in liver, brain, lung, spleen, and
testis DNA, and in hemoglbbin and plasma proteins (41). Vhen the dose-
response was determined for liver DNA, hemoglobin, and plasma proteins, all
were found to be non-linear.
HYDROXYLAMINE
4
In a report that described the mutagenicity of N -hydroxylcytidlne, It was
stated that N -hydroxycytldlne was formed through the reaction of DNA with
hydroxylamlne (100).
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DIMETHYLCARBAMYL CHLORIDE AND DIETHYLCARBAMYL CHLORIDE
Dlmethylcarbamyl chloride (DHCC) and diethylcarbamyl chloride (DECC) are
Industrial Intermediates that are use In the production of pharmaceuticals and
carbamate pesticides. DMCC Is a strong rat carcinogen whereas DECC Is a weak
carcinogen. When DllCC and DECC were reacted with calf thymus DNA lri vitro,
several adducts were formed; 6-dimethyl-carbamyloxy-2'-deoxyguanosine, 6-
dlethylcarbamyloxy-2'-deoxyguanoslne, 4-dlmethylamlno-thymidine, and 6-
dlmethylamlno-deoxyguanoslne (191).
ALKYLATING AGENTS
There 1s a large class of compounds that either directly or through
metabolic activation alkylate DNA. The vast majority of these compounds add
methyl or ethyl groups to DNA purines and pyrlmidlnes. Several excellent
reviews have been written on this class of compounds (40, 58, 101, 148-150,
196, 197, 198), and they should be consulted for more detailed information.
The literature summary will be subdivided into specific subclasses of
alkylating agents.
ALKYL SULFATES
DIMETHYL SULFATE
Alkyl sulfates are industrial chemicals that are commonly used alkylating
reagents. The two most commonly used alkylating agents are dimethyl sulfate
and diethyl sulfate. Uhen reacted with calf thymus DNA In vitro, dlmethy^
sulfate formed the following adducts: 3-methylguanlne, 7-methylguanine,^0 -
methylguanlne, 1-methyladenine, 3-methyladenlne, 7-methyladenlne, a^d 0 -
methylcytosine (156). The same adducts were detected (except for 0 -methyl-
cytoslne) when cell cultures were Incubated with dimethyl sulfate (4^,79,145).
The levels of three adducts (7-methylguanlne, 3-methyladenine, and 0 -methyl-
guanlne) in V79 cells treated with 8 and 15 ppm of dimethyl sulfate were
measured. The levels of adducts Induced at 15 ppm were 92.4, 12.0, and 0.5
umol/mol DNA (145). Only 7-methylguanlne levels appeared to be directly
related to the concentration of dimethyl sulfate. Also, the half-lives of two
adducts were determined in the V79 cells. 7-Methylguanine and 3-methyladenlne
had half-lives of 14 and 4 hours, respectively (46).
DIETHYL SULFATE
Diethyl sulfate also forms adducts with DNA. When diethyl sulfate was
reacted with DNA In vitro, the following adducts were formed: 1-, 3- and 7-
ethyladenine, 3- and 7-ethylguanine, and 3-ethylcytosine (196).
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Aim ALKANE sulfonates
HETHYLMETHANESUL FOMATE
Alkyl alkane sulfonates are research compounds used for alkylation
reactions. The primary compounds that have been studied are methylmethane-
sulfonate (MNS) and ethylmethanesulfonate (EMS). MMS formed 3-methyladenlne
and 7-methylguanlne adducts In Chlamvdomonas relnhardl (207). The half-lives
of the adducts were about 2 and 10 hours, respectively. The half-lives are
very Blmllar to those measured in V79 cells treated with dimethyl sulfate
(46). When MMS was administered to female rats, both DNA and protein adducts
were detected. After administration of SO mg/kg of MMS, 12 ug of 7-methyl-
guanlne waa excreted In the urine over 24 hours (64). The animals were
sacrificed 7 days after dosing, and the hemoglobin level of S-methylcystelne
attributable to the MMS was about 37 ng/mg hemoglobin.
ETHYLMETHANESULFONATE
When DNA was exposed to EMS, the following adducts were formed: 7-ethyl-
guanlne, 3-ethylcytoslne, 3-ethyladenlne and 1-ethyladenlne (196). EMS also
forms DNA adducts in vivo. Uhen tfistar rats were administered 200 mg/kg EMS,
7-ethylguanlne, 3-ethylguanlne, 3-ethyladenlne, and 0 -ethylguanlne were
detected in rat liver (57). The persistence of certain adducts were then
measured. The half-life of 7-ethylguanlne was about 6 days, and the half-
lives for both 3-ethylguanlne and 3-ethyladenlne were less than 6 days. The
exposure-dose relationship was explored in a study that exposed Neurospora
crassa and Saccharomvces cerevlslae to EMS. The two strains were exposed to
EMS concentrations from 2.5 to 50 mM. Ethylatlon (unspecified adducts) of DNA
was linear but was less than proportional to the^concentration of EMS (221).
Ethylatlon ranged from 0.8 to 8.6 ethylat^ons/10 nucleotides In Neurospora
crassa and from 0.6 to 6.7 ethylatlons/10 nucleotides in Saccharomyces
cerevlslae.
n-BUTYLMETHANE SULFONATE
The alkyating potential of n-butylmethanesulfonate has
tfhen reacted with DNA in vitro, the following adducts were
butylguanosine, 7-n-butylguanoslne, and 3-n-butylguanoslne
DIALKYL NITROSAMINES
N-NITROSODIMETHYLAMINE
Dialkylnitrosamines are a group of chemicals used widely In Industry and
In cancer research. N-Nitrosodlmethylamlne (DMN) Is used in Industry as an
antioxidant, as an additive for lubricants, and as a softener of copolymers.
N-Nltrosodlethylamlne (DEN) has similar uses and Is also used as a gasoline
additive. Two animal studies were locat|£ on DMN. DMN was administered to
¦Ice, and adducts were detected vla,the P-postlabellng assay. The adducts
detected were 7-methylguanlne and 0 -methylguanlne (182). In another study,
DMN (10 mg/kg) was administered to Wlstar rats, and the following adducts were
detectedi 7-methylguanlne (379 umol/mol DNA-P), 3-methylguanine (2 umol/mol
been investigated,
formed: 06-n-
(187).
39
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DNA-P), 77methyladenlne (<1 umol/mol DNA-P), 3-methyladenine (9 umol/mol DNA-
P), and 0 -methylguanine (37 umol/mol DNA-P) (57). The half-lives of all of
the adducts were less than 6 days.
N-NITROSODIETHYLAMINE
OEN was also administered (50 mg/kg) to Ulstar rats. The adducts
dectected were 7-ethylguanlne (31 umol/mol DNA-P^, 3-ethylguanine (3 umol/mol
DNA-P), 3-ethyladenine (4 umol/mol DNA-P), and 0 -ethylguanlne (2 umol/mol
DNA-P) (57). Once again, the half-lives of all of the adducts were less than
6 days. Angther experiment expgsed rat hepatocytes to 40 ppm DEN, and the
levels of 0 -ethylguanlne and 0 -ethylthymine were measured at 8, 16, and 28
days. The levels of the ethylguanlne decreased from 0.3 to less than 0.2 pmol
adduct/umol guanine whereas the levels of ethylthymine increased from 5 to 10
pmol adduct/umol thymine (184). The authors postulated that the ethylated
thymine adduct levels might be an important part of DEN-induced
carcinogenicity.
MISCELLANEOUS NITR0SAMINES
DNA adduct information was located on four other nitrosamlnes. N-
Nitro8omethylbenzylamine (MBN) is a research chemical that was administered to
Vistar rats. Four hours after an intravenous injection of MBN (0.017
mmol/kg), 7-methylguanine was detected in the esophagus, liver, lung, and
forestomach, with the highest levels being in the esophagus (344 nmol/mol
guanine) (94). N-Nitroso-N-acetoxymethyl-N-2-oxopropylamine (NAM0PA) is a
model nitrosamine that is used to mimic the believed active form of N-nltroso-
dipropylamlne in rats. Vhen subjected to enzymatic hydrolysis in the presence
of DNA, the following adducts were detected: 0 -methylguanine, 7-methyl-
guanine, and 3-methyladenine (131). Another research chemical, nitrosobis-(2-
hydroxypropyl)amine induces liver tumors in hamsters but not in rats. Uhen
this compound was administered to rats and hamsters (20 mg/anlmal), more
agducts were found in hamster DNA (134). The levels of 7-methylguanlne and
0 -methylguanine in hamster liver DNA were about 280 and^23 dpm versus about
40 and 0 dpm in rat liver DNA. Finally, the levels of 0 -methylguanine in the
rat Induced by the tobacco carcinogen 4-(methylnitrosamino)-l-(3-pyridyl)-l-
butanone (NNK) were measured by using polyclonal antibody techniques. F344
rats were injected 60 tines (40 mg/kg) over the course of 20 weeks and nasal
mucosa, lung, liver, kidney, esophagus, spleen, and heart DNA was analyzed for
0 -methylguanine. Measurable levels were found in the nasal mucosa (8
umol/mole guanine) and lung (11 umol/mole guanine) (42). If the rats were
only given one injection (87 mg/kg), adduct levels were measurable in the
nasal mucosa (219 umol/mol guanine), lung (13 umol/mol guanine), and liver (34
umol/mol guanine).
Many carcinogens have been associated with tobacco smoke, such as 5-
methylchrysene, benzo(a)pyrene, and 13 N-nltrosamlneB. However, In snuff, N-
nitrosamlnes are the only carcinogens found In significant amounts. The
ability of two tobacco-specific N-nltrosamlnes [N'-Nltrosonornicotlne (NNN)
and 4-(methylnitrosamino)-l-(3-pyridgl)-l-butanone (NNK)] to alkylate DNA was
studied. NNK, but not NNN, formed 0 -methyl-guanlne when the compounds were
administered to rats (42).
NNN forms a nornlcotine-DNA adduct rather than a simple methyl group.
40
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M-HTTROSOUREAS
N-nitrosoureas are research chemicals and therapeutic agents that alkylate
DMA. The vast body of ONA adduct research done with N-nitrosoureas has
utilized the compounds H-methylnitrosourea and N-ethylnitrosourea.
N-METHYL NITROSOUREA
N-Methylnitrosourea (MNU) forms DNA adductg both i£ vitro and in vivo.
When calf thymus DNA w^s incubated with MNU, 0 3-, 7-methylguanlne, 1-, 3-,
7-methyladenine, and 0 -methycytosine were detected (156). Some or all of
these adducts have also been found in cells treated in culture (46,71,79,156),
rats (65, 107), and mice (79).
Two limited dose-response studies were located. V79 cells werg treated
with 0.08, and 0.40 mM MNU and adduct levels of 7-methylguanine, 0 -methyl-
guanlne, and 3-methyladenine were measured 2 hours after incubation with HNU.
The levels of 7-methylguanine (17.4 and 89.3 umol/mol DNA-P), 0 —methylguanlne
(2.1 and 11.7 umol/mol DNA-P), and 3-methyladenine (0.6 and 2.9 umol/mol DNA-
P) were roughly proportional to the concentration of MNU present (46).
However, another dose-response study with V79 cells did not show complete
proportionality. The cells gere treated with 30 or 60 ug/ml MNU, and the
levels of 7-methylguanine, 0 -me£hylguanine, and 3-methyladenine were
measured. 7-Methylguanine and 0 -methylguanlne levels showed a proportional
relationship with dose (50-102 and 7.5-14.4 umol/mol DNA-P, respectively), but
3-methyladenine levels did not (5.4-6.2 umol/mol DNA-P) (156).
Several studies have looked at the persistence of MNU-induced adducts.
Although most adducts are removed rather quickly, some do appear to persist.
Chinese hamster cells were Incubated glth MNU, and the adduct levels of 3-
methyladenine, 7-methylguanine, and 0 -methylguanlne were measured at 0 and 20
hours after incubation. The levels of 3-methyladenlne and 7^methylguanlne
dropped 84 and 55 percent, respectively, but the levels of 0 -methylguanlne
adducts had not decreased at all (71). When |emale rats were treated with
MNU, seven.adducts were identified: 3-, 7-, 0 -methylguanlne, 1-, 3-methyl-
adenine, 0 -methylthymine, and an imidazole ring-opened 7-methylguanine (107).
With the exception of 0 -methylguanlne and the imidazole ring-opened 7-methyl-
guanine, the half-lives of the adducts were all under 2 days. The half-life
of 0 -methylguanlne was about 3 days, and the half-life of the Imidazole ring-
opened adduct was greater than 21 days. The authors suggested that the ring-
opened adduct might be important in the process of tumorlgenesis because of
its extreme persistence. Another study looked at the persistence of 7-methyl-
guanlne and 06-methylguanine and their relationship to susceptibility to MNU-
induced tumorigenesis. Five strains of mice with widely varying
susceptibilities to MNU-induced tumorigenesis were administered MNU. The
absolute levels and half-lives of the two adducts were not appreciably
different in any of the mouse strains (79). The author postulated that the
levels of adducts and their persistence was not necessarily predictive of
susceptibility to tumorigenesis. Another explalnatlon might be that they were
looking at the wrong adducts. Perhaps levels of the imidazole ring-opened 7-
methylguanine adduct would have correlated with susceptibility.
41
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N-ETHYLNITROSOUREA
H-Ethylnitrosourea reacts with DNA vitro and lr* vivo. When ENU was
Incubated with calf thymus DNA, the following adducts gere detected: l-j 3-,
7-ethyladenine, 3-, 7-ethylguanine, 3-ethylcytosine, 02*-ethylguanine, 0^-ethyl
cytosine, 0 -et^yluracil, ang 0 -ethyluracil (196). 0 -ethylthymine, 0 -
ethylthymine, 0 -cytosine, 0 -ethylguanine, 7-ethylguanine, and 3-ethyladenine
were detected in rat brain, liver, spleen, intestine, muscle, kidney, lung,
and also in human fibroblasts (196).
Two studies were located that explored the dose-response relationship of
ENU-induced adduct formation. Rats were Injected with H-ENU (10 or 100
mg/kg) and the levels of 0 -ethylguanine, and 7-ethylguanine were measured 1
hour later in testis DNA. The levels of 7-ethylguanine increasgd as expected
(1.79 and 17.3 umol adduct/mol guanine) whereas the levels of 0 -ethylguanine
actually increased more than expected (1.31 and 18.1 umol adduct/mol guanine)
(190). The authors attributed the greater than proportional adduct increase
to the saturation of alkylation of selective protein sites at the higher ENU
concentration. Another study measured the dose-response relationship of ENU
in vitro and in vivo by using an immunoassay system. ENU reacted with DgA
linearly over the concentration range of about 0.1 to_^00 ppm yielding 0 -
ethylguanine concentrations ranging from about 6 x 10 to 1 x 10 mol
adduct/mol guanine (153). Also, trgatment of rats with 3 to 100 mg/kg ENU
resulted in a linear formation of 0 -ethylguanine from 0.3 to 30 umol
adduct/mol guanine (153).
The half-lives of the adducts varied considerably. As was the case with
MNU, 3-ethyladenine and 7-ethylguagine adducts were removed efficiently by
Chinese hamster ovary cells, but 0 -ethylguanine adducts were not (71). In a
study that measured the half-lives of 0- and N- ethyl adducts in mammalian
cells and in the rat, all of the half-lives were about the same; 30 to 60
hours (196). The only exception was 3-ethyladenine; its halfllfe was about 8
to 10 hours.
MISCELLANEOUS NITROSOUREAS
n-Propylnitrosourea has been reacted with calf thymus DNA, and
Interestingly, two types of adducts werg found: not only the expected n-
propyl adducts, 7-n-propylguanine and g -n-propylguanlne, but also the lso
derivatives, 7-lso-propylguanlne and 0 -lso-propylguanine (151). During the
reaction with calf thymus DNA, the propyl carbonlum ion likely rearranges to a
more stable electronic configuration that would lead to the formation of
isopropyl adducts. n-Butylnitrosourea was reacted with calf thymus DNA, and
both n- and iso-butvl adducts were formed (187).
The research chemical, N-(2-oxopropyl)-N-nitrosourea wgs reacted with calf
thymus DNA, and the following adducts were detected: 7-, 0 -methylguanine, and
3-methyladenine (131).
Several compounds In this class are bifunctlonal alkylating agents;
bls(chloroethyl)nitrosourea (BCNU), bls(fluoroethyl)nltrosourea (BFNU), and N-
(2-chloroethyl)-N'-cyclohexyl-N-nitro80urea (CCNU). These compounds are used
In the treatment of various cancers, and they appear to kill cancer cells by
inducing lntrastrand DNA cross-links (101).
42
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MITROSOGUANIDTNES
Reports were located on the alkylating ability of N-methyl-N'-nitro-N-
nltrosoguanidige (MNNG) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). MNNG
forms 7- and 0 -methylguanlne adducts, and ENNG^forms 3-ethylade^ine, 3-, 7-
ethylguanln^, 3-ethylcytosine, ^-ethyluracil, 0 -ethylguanine, 0 -ethyl-
cytoslne, 0 -ethyluracil, and 0 -ethyluracil (26,196).
MISCELLANEOUS NITROSO COMPOUNDS
1-NITR0S0-5,6-DIHYDROURACIL AND N-NITROSO-N-METHYLURETHANE
The anticancer agent and rat liver carcinogen, l-nltroBO-5,6-dihydrouracil
is activated to form the electrophillic species Z-carboxyethylcarbonium ion.
This species reacts with DNA to form the adduct 7-(2'-carboxyethyl)guanine
(146). When rats and hamsters gere given nitroso-2,6-dimethylmorpholine, two
adducts were detected; 7- and 0 -methylguanlne (134). The levels in the
hamster (the susceptible species) were more than 10 times higher than those^in
the rat. Finally, N-nitroso-N-methylurethane was reported to form 7- and 0 -
adducts in vitro (26).
HYDRAZINE DERIVATIVES
HYDRAZINE
Hydrazine derivatives occur both as synthetic and natural products
Including sources of food. Hydrazine, which dges not contain any carbon
atoms, induces DNA methylation. Both 7- and 0 -methylguanlne adducts have
been detected (101).
DIMETHYLHYDRAZINE
1,2-Dimethyl hydrazine (SDMH) is a potent colon carcinogen £n rats and
mice. Three agducts have been detected in these species; 7-, 0 -methyl-
guanlne, and 0 -methylthymlne (27,91,99,184). Several studies have been
conducted to determine If there is a relationship between DNA adduct levels
and the carcinogenic activity of SDMH. SDMH was administered to two strains
of rats, ICR/Ha (susceptible) and C57BL/Ha (resistant). Although a good
correlation existed between adduct levels in particular segments of the colon
and the incidence of cancer in the susceptible strain, no correlation was seen
in the resistant strain (99). In another study, rats were injected weekly
with SDMH, and the levels of 7- and 0 -methylguanlne were monitored 1 week
after each injection. No adducts were detected in the colon; only 7-methyl-
guanlne was detected in the liver, and both adducts were detected in the
kidney (91). This does not correlate well with the fact that SDMH is
primarily a colon carcinogen. The authors postulated that the rate of cell
replication was an Important factor in SDMH-induced carcinogenicity.
43
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SDMH can Induce liver tumors but only In hepatocytes and not in non-
parenchymal cells £NPC). Rats were administered SDMH In drinking water' (30
ppn), and 7- and 0 -methylguanlne levels were monitored In hepatocytes and
HPC's. Levels of 7-methylguanine^were about the same in both cell types over
the 28 day study, but levels of 0 -methylguanlne were essentially 0 In
hepatocytes and were found in NPC's at a concentration of 20 pmol/mg ONA (27).
Similarly, administration of SDMH led to a increase.in cell replication in
NPC's but not in hepatocytes (132a). In addition 0 -methylthymine might be an
Important adguct in ^DMH-induced carcinogenesis. When rats^were administered
SDMH, both 0 - and 0 - adducts w^re formed. Although the 0 - adduct levels
w^re initially higher than the 0 - levels, the increased persistence of the
0 - adduct led to an increased relative level of the 0 - adduct over time
(184). Many dlfferent^ethodologies were used ln the above studies, including
Immunoassay (184) and P-postlabeling methods (182).
HALOGENATED ALKYLATING AGENTS
The following list of chemicals are grouped together because of the
presence of halogen atoms. Only one or two papers were located on each
compound.
1,2-DIBR0M0ETHANE AND 1-BR0M0,2-CHL0R0ETHANE
1,2-Dibromoethane is used as a soil, grain, and fruit fumigant, as an
industrial solvent, and as a lead scavenger in gasoline. 1,2-Dibromoethane is
a mutagen and is capable of producing liver, lung, stomach, mammary, adrenal,
skin, spleen, and kidney tumors. 1,2-Dibromoethane was reacted with DNA in
vitro, and it was determined that the rate of reaction with DNA was greatly
increased in the presence of glutathione s-transferase (GSH s-transferase)
(160). The same enhancement was seen when 1-bromo,2-chloroethane was
Incubated with DNA and GSH s-transferase. It is believed that the
electrophillic species that reacts with DNA is the S-(2-bromoethyl)GSH
eplsulfonium ion, and the adduct formed has been identified as S-[2-(7-
guanyl)ethyl]GSH. Vhat makes these compounds particularly interesting is the
fact that GSH is Involved with bioactivatlon of a carcinogen rather than with
its usual role of detoxification.
EPICHLOROHYDRIN
Epichlorohydrin, l-chloro-2,3-epoxypropane is a solvent for natural and
synthetic resins, gums, cellulose esters and ethers, paines, varnishes, nail
enamels and lacquers, gnd cement for Celluloid. Epichlorohydrin has been
shown to form 7- and 0 -alkylguanlnes (26).
VINYL CHLORIDE
Vinyl chloride is an important industrial chemical that is used in the
production of polyvinylchloride (PVC). Vinyl chloride is also a carcinogen.
Its active form is chlorooxlrane (i.e., ethylene oxide with an attached
chlorine) (177a). It is both a mono- and blfunctlonal alkylating agent,
attacking primarily adenine, cytosine, and guanine (177a, 196).
44
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MISCELLANEOUS ALKYLATING AGENTS
The compounds described below are of a diverse nature, and only one or two
literature citations were located on each one.
DICHLORVOS AND METRIFONATE
The organophosphorus pesticide, dichlorvos, was fed to rats, and the DNA
from the lung, liver, heart, brain, kidney, testis, and spleen was isolated
and pooled. The adduct 7-methylguanine was detected (236). Similarly, the
pesticide metrlfonate was administered intraperitoneally to rats. 7-Methyl-
guanlne was found In the liver and kidney. Maximal levels were acheived six
hours after administration, and depending on the dose, the half-life of the
adduct was 5 to 15 hours (53).
ETHIONINE
Ethionine is a carcinogen that was shown to bind to DNA, RNA, and proteins
in the rat, but no information was given on specific adducts (130).
GLYCIDALDEHYDE
Glycldaldehyde, 3-epoxy-l-propanal, has been shown to alkylate guanine
at positions 7 and 0 (26).
GYROMITRIN
Gyromitrin (acetaldehyde N-methyl-N-formylhydrazone) is found in the false
morel mushroom. Vhen administered to rats by gavage, it was found that liver
but not lung DNA contained 7-methylguanine (86).
1,3-PR0PANE SULTONE AND BETA-PROPIOLACTONE
1,3-Propane sultone and beta-propiolactone are both strained cyclic
compounds that can alkylate^DNA. When 1,3-propane sultone was reacted with
DNA in vitro. 1-, 7-, and 0 -alkylguanine derivatives were detected £88). In
another in vitro study, beta-propiolactone formed alkylated 7- and 0 -alkyl-
guanine adducts (26).
STREPT0Z0T0CIN
Streptozotocin is an antibiotic used for the induction of diabetes in
experimental animals and as a therapeutic agent in the treatment of pancreatic
neoplasms. Vhen Streptozo-toxin was administered to rats (21 mg/kg), the
following adducts were formed: 3- and 7-methyladenlne and 7- and 0 -methyl-
guanine (34). The adducts were measured In the liver, kidney, intestine,
brain, and pancreas. Adducts levels ranged from less than one pmol/umol
guanine to 486 pmol/umol guanine.
45
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RECOMMENDATIONS FOR FUTURE RESEARCH
In the previous sections of this report, discussions have been presented
on methods for measuring carcinogen adductB, on biological fluids and tissues
that can be used to monitor adduct formation, and on compounds that have been
studied that form adducts. From the information presented, it is possible to
propose candidate compounds for further study. It is also possible to propose
detection methodology and biological samples suitable for large scale
population monitoring. However, these tools alone are not sufficient for the
interpretation of the data that would be obtained from such a study. Much
more preliminary experimental data is required before such epidemiological
data could be Interpreted.
Figure 6 shows a diagram that traces the path of a chemical carcinogen
from Its presence in the environment to the onset of cancer. The main
objective of monitoring human subpopulations for carclnogen-ONA adduct
formation Is to obtain a better estimate of the dose than is obtained from
exposure data that is currently being obtained. A further objective of
blomonltorlng is to obtain better estimates of risk by monitoring specific
damage caused by carcinogens. In reviewing Figure 6, it can be seen that both
DNA adducts and protein adducts would provide a better estimate of dose and of
risk because the adduct levels measured are obtained from the organism (man)
in which one wishes to estimate dose. However, carclnogen-macromolecule
adducts are not a panacea. Adduct levels, In most cases, cannot be plugged
Into a simple equation to receive the answer (dose levels in man). Many steps
In the process from compound uptake to the onset of cancer are either poorly
understood or extremely complicated.
The dose response of many carcinogens has been studied in experimental
animals, and almost all show at least some linearity in the dose response
curve. However, much research still remains to be done. Most studies have
used concentration ranges of only one or two orders of magnitude, and the
dosing levels were very high (to facilitate adduct detection). Very little is
known about dose response at very low levelB of carcinogens. Any compound
that is chosen for a human study should have animal experimental data that
shows the dose response at very low levels since this is the level at which
most exposures are likely to occur. Another problem with most present animal
studies is the method of dosing. Most studies dosed animals by administering
a single dose of the carcinogen being studied. In a real life situation,
dosing would very likely be semi-continuous and perhaps intermittent.
Additional animal research needs to be conducted so there will be a better
understanding of dose-response under these dosing conditions.
The second step in the process from exposure to onset of disease is
metabolism and distribution in the body. Metabolism Is not as much of an
issue with direct-acting carcinogens because the compounds form adducts
without requiring metabolic activation. However, metabolism can still
inactivate direct acting carcinogens. The manner in which the compounds are
distributed throughout the body is important In determining what tissues are
exposed to adduct-formlng compounds. Animal studies that have measured the
toxicokinetics of these compounds can be used to estimate how compounds are
distributed in the body, so it should be possible to make reasonably accurate
estimates of the dose received by an individual if protein adducts are
quantified. The estimation of dose received of an indirect-acting carcinogen
46
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Is not as easy. In order to make a reasonable estimation of the dose received
over the period of exposure measured by protein adducts, one needs information
on the level of metabolic activity in the individual from which the samples
are taken. The measurement of enzyme levels will be difficult, as the highest
concentration of most xenoblotic-metabolizlng enzymes Is found in the liver.
Liver biopsies are out of the question in most cases, so estimations of enzyme
activity will have to be made via other means. Perhaps the measurement of the
levels of endogenous metabolites will make It possible to estimate enzyme
levels.
The use of carclnogen-DNA adducts as a measure of dose received la likely
to be less accurate than the use of protein adducts. The main reason for the
decreased accuracy of DNA adducts as a measure of exposure is that the adduct
levels are altered via DNA repair and dilution through cell division. Protein
adducts also decrease with time, but the decrease can be accurately estimated
because the half-lives of the proteins are accurately known. There is much
less known about DMA repair, and hence, the uncertainty associated with dose
would be greater.
DNA adducts would be much better to use as an estimate of cancer risk
because one is measuring the actual damage to the cellular genetic
Information. However, the levels of DNA adducts would at best provide a rough
estimate of risk for the following reasons. Prior damage to DNA might not be
measurable as DNA adducts because the adducts may have already induced a
mutation. The mutation would not be measurable as an adduct, so past DNA
damage could be hidden. Also, not all adducts are associated with increased
incidence of cancer. Research must be conducted to determine what adducts are
associated with increased cancer risk. Finally, very little is known about
how mutations trigger the onset of cancer. Optomistically, it will be several
years before the progression from mutation to cancer onset.
The preceding has pointed out some of the inadequacies in present
knowledge of the relationship between dose received, carcinogen-macromolecular
adduct formation, and the onset of disease. Although much research still
needs to be conducted before the measurement of adducts would be useful, the
potential for their use in exposure assessment is great. The measurement of
carcinogen-macromolecule levels in an organism for dose estimation is
inherently more accurate than the measurement of carcinogen levels in the
environment.
RECOMMENDATIONS FOR TISSUES AND FLUIDS TO BE ANALYZED
There are two main points to be considered when choosing fluids or tissues
to detect adduct formation. The first point is one of invasiveness. If the
sample collection technique is too Invasive, it will be impossible to obtain
enough volunteers for the studies. The second point involves the usefulness
of the sample. The further away the sample is from the target organ, the less
representative the measured adduct formation will be of the adduct levels at
the target site unless animal studies indicate otherwise. Therefore, tissue
and fluid selection will be a compromise between these two points.
The best compromise fluid is blood. Sample collection is relatively non-
invasive, and it contains several molecules that can contain covalent adducts.
47
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White blood cells contain DMA; therefore, DNA adduct formation can be
monitored. However, one must remember-that the DNA adduct level may not
correlate with the DNA adduct level In the target organ(s) or with the dose
received. In addition, protein adducts can be monitored. The two main
proteins to be monitored would be hemoglobin and human serum albumin. Each
protein would give different levels of Integration data as the half-lives of
the proteins are 120 and 20 days, respectively. Also, human serum albumin is
extra-cellular and is not protected by a cellular membrane; it might show a
higher level of protein adduct formation. In summary, both integration and
equilibration data can be obtained from blood.
Urine is also an excellent choice for monitoring DNA adducts. When DNA
repair occurs, the removed adducts are excreted in the urine. However, not
many carcinogen adducts have been monitored in the urine, so additional animal
studies would have to be conducted. In one animal study, the levels of
aflatoxln-DNA adducts found in the urine correlated with the administered dose
of the carcinogen. Theoretically, any adduct that is repaired could be
monitored by using this fluid.
Tissues obtained during a biopsy or autopsy would provide the most
accurate information concerning the level of damage that occurs at the target
site. The problem with biopsy samples Is that they are very difficult to
obtain, and it would be difficult to obtain volunteers for such a study. The
question that needs to be asked is if target DNA adduct level information is
really needed. If the main purpose of the planned studies is to obtain dose
information, that is, levels of the carcinogen that have entered the body,
then the information obtained from adducts measurable in blood may be
sufficient.
In summary, the best approach to monitoring human subpopulatlons would
entail collection of both blood and urine samples. This approach would allow
the collection of the most data with a minimum of discomfort to the
individuals.
SUGGESTED MEASUREMENT TECHNIQUES
Biomonltoring Implies the collection of several biological samples over a
period of time from many individuals. As a result, many samples will have to
be analyzed. Also, exposures to the carcinogen(s) of interest will normally
be at extremely low levels, bo the adduct levels will likely be at or below
the detection limit of most methods. Because the adduct levels will be very
low, interferences may present a serious problem. The above facts require
that the methods be Inexpensive, fast, very sensitive, and selective. Of all
the adduct detection methods available, only t^ appear to lie within the
above constraints: Immunoassay techniques and P-postlabellng techniques.
The Immunoassay technique, except for the initial antibody Identiflcatl^*
isolation, and preparation, is the simpler of the two techniques. The P-
postlabellng technique, although more cumbersome, can be more sensitive. A
possible method to monitor for DNA adducts w^ld Include a rapid screening
with the Immunoassay method followed by the P-postlabellng method for thoBe
that showed up negative on the Immunoassay screening. This procedure would be
the most cost effective as only those samples wl^ very low levels of DNA
adducts would be analyzed by the more expensive P-postlabellng method.
48
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SUGGESTED COMPOUNDS FOR BIOLOGICAL MONITORING
Biological screening of large populations to determine the presence of DNA
adducts will likely prove very expensive, no matter how inexpensive assays
are. The process of tracking large numbers of people Is very expensive in and
of Itself. Therefore, compounds selected for monitoring should be those for
which there is a high expectation of demonstrating a useful exposure-dose
relationship cost effectively. An economic criteria that should also be
Included is the presence of relevant preliminary research. A lot of
preliminary animal data will be needed before a pilot human study can be
conducted. If a large part of data needed has already been published on
particular compounds, serious consideration should be given choosing one of
them. However, there are probably few, if any, compounds that have a large
part of the preliminary research already completed.
An Important criteria to be considered in the selection of a compound for
a pilot study is the existence of exposed populations. Coupled with this
would also be the criteria that an unexposed population also exists. For
example, studies have been conducted where populations were monitored for the
presence of benzo(a)pyrene adducts. The studies showed that benzo(a)pyrene
was so ubiquitous in the environment that adduct levels above background were
only detected in persons exposed to high levels of the compound. In other
words, the background levels of benzo(a)pyrene-DNA adducts were so high that
they masked any dose-response curve that was attributable to occupational
exposure.
The EPA has a list of priority compounds that should be considered for
further study with this technique of exposure assessment. It would benefit
the Agency If one of the priority compounds were chosen. However, the
presence or absence of a compound on the list should not be the overriding
consideration. The primary consideration should be a compound that has the
highest expectation of producing a usable exposure-dose relationship.
Therefore, human population studies in which information on exposure is
already quantitatively known might prove useful to determine the feasibility
of using carcinogen-macromolecule adducts to monitor exposure to genotoxic
chemicals. An Ideal population for an initial study would be patients
receiving anticancer drugs.
For example, the Health Effects Research Laboratory (HERL) In the Research
Triangle Park Is conducting an exposure-dose relationship study with 2,5-
diazeridinyl-3,6-bicarboethoxyamino-l,4-benzoquinone (AZQ). AZQ Is a
chemotherapeutlc agent for brain tumors. Unfortunately, long term studies are
difficult because of the extremely poor prognosis for patients with brain
tumors.
Another chemotherapeutlc agent, clsplatin, would be ideal to research the
exposure-dose relationship. Current research indicates that this therapy is
effective in about 50 percent of patients receiving this treatment. In those
patients where treatment is effective, DNA adducts are detected in circulating
white blood cells, whereas adducts were not detected in patients resistant to
clsplatin treatment. This suggests that there are factors that Influence the
levels of clsplatin in the vicinity of DNA, such as drug metabolizing enzyme
49
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levels, glutathione levels, or permeability of the membrane to cisplatin.
This population would provide an ideal setting to determine if a blomarkers
package can be developed to correct for individual variation in the above
factors. This study might involve the monitoring of (1) DNA adducts in white
blood cells or the target organ (biopsy), (2) DNA adduct levels in urine to
estimate DNA repair, (3) Thioether compounds in urine to monitor glutathione
levels, (4) glutathione levels in the blood, and (5) cisplatin-protein (Hb and
serun albumin) adducts in the blood. Also, i_n vitro studies of cancer cells
In culture might also be used to estimate rates of DNA repair or to measure
cell membrane permeability to cisplatin. This list of variables to monitor is
by no means complete and is given only to suggest some variables that may be
Important. This study to look at a biomarkers package with patients receiving
cisplatin will not only test the potential of using a biomarker package to
measure exposure but should also lead to a more effective chemotherapeutic
treatment. It is proposed that a study of this nature be used to support the
Office of Research and Development (ORD) rotational assignment or sabatical
programs under the aegis of the EPA in order to improve the scientific quality
of personnel, and to give personnel the opportunity to participate in research
and to contribute to publications. Such an agreement could be established
with NCI, Bethesda, MD, where research on DNA adduct measurements in relation
to cisplatin is currently being conducted. NCI has expressed interest in
conducting a study of this nature if the appropriate manpower can be supplied.
The analysis of alkylated DNA and protein adducts may be a useful
monitoring project. Alkylating agents are widely distributed compounds, and
they have been detected in some foods or food byproducts as well as in tobacco
products. In the past, there has been some debate as to whether these
compounds are a threat to the general population because of their short half-
life in the body. It is this short half-life that also makes the measurement
of the free compound in tissues or fluids impossible (42). Therefore,
measurement of adducts may provide a method for estimating the dose of
alkylating agents received by individuals. A population of exposed
individuals is readily available: persons who use tobacco products.
Estimating exposure to specific alkylating agents present in tobacco products
should be fairly simple because the concentration of alkylating agents in the
tobacco and the amount of tobacco product used can be measured easily. In
addition, the dosing regimen is relatively consistent, as tobacco usage would
occur on a more or less regular basis. Smokeless tobacco may be a good choice
because levels of the alkyating agents 4-(methylnitrosoamino)l-l-(3-pyridyl)-
1-butanone (NNK), and N-nitrosonornicotlne (NNN) are very high and because
pyrolysia-produced carcinogens such as benzo(a)pyrene, 5-methylchgysene, and
dlbenz(a,h) anthracene are not present. An antibody exists for 0 -methyl-
guanine, and it has been used in at least one study to measure DNA damage
induced by activated NNK (42). The use of this antibody may be complicated
because it showed cross-reactivity with other methylated bases and some cross
reactivity with native guanine. Although the cross-reactivity with native
guanine is low, it would be an interference if the levels of the methylated
guanine that are being measured are low. This problem can be overcome by
using high performance liquid chromatography to remove the unmodified guanine
residues from the sample. Of course, this will add to the cost of each
Although more difficult, methylated bases can be monitored by using the P-
postlabellng technique, and low detection limits should be possible. NNN
would also be a good tobacco-derived alkylating agent to measure because the
NNN-DNA adduct can be easily attributed to tobacco. NNK-derlved DNA adducts
are the same as any other methylating agent. Therefore, methylated DNA cannot
50
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be attributed to any specific methylating agent.
One compound that may be a good choice Is the mycotoxln aflatoxln-B,.
Various levels of aflatoxln exposure exist. Corn and peanut farmers ana
handlers would likely be more highly exposed than others, and the general
population would be exposed through peanut and corn food products. A problem
that might exist would be the lack of negative controls. If all individuals
have a high background level of aflatoxln B^ adducts, then a reasonable dose-
response relationship may not be found. It would be very useful If an
exposure-dose relationship can be demonstrated because analytical
methodologies for measuring levels of aflatoxln In the environment are either
Inaccurate or very complex. Excretion of aflatoxln-DNA adducts has been
detected In laboratory animals that were administered aflatoxln, and a useful
exposure-dose relationship was established. Also, the l^ge, hydrophobic
adduct produced by aflatoxln is amenable to analysis by P-postlabellng
methods so that low levels of adducts could be detected.
Other compounds have been located that did not have many references on
adducts in the computerized computer search but probably should be given
consideration for further study. Vinyl chloride is an industrial chemical
that about three million workers are exposed to. Its primary use is in the
manufacture of polyvinyl chloride (PVC). Vinyl chloride binds to hemoglobin
when administered to rats (64); this suggests that vinyl chloride may also
form adducts with DNA. Other chemicals in the polymer industry might also be
good future candidates for study such as acrylonitrile, epichlorohydrin, and
styrene. Styrene (In the form of styrene oxide) has been shown to form DNA
adducts, and the pertinent literature has already been summarized. The
Industrial Intermediate dlmethylcarbamyl chloride, used in the production of
pharmaceuticals and carbamate pesticides, might also be of interest. One
might also look at methylated adducts formed by the pesticides dichlorvos and
metrlfonate. However, both pesticides methylate DNA, so the adducts would not
be directly traceable back to the pesticides. o-Toluidine hydrochloride Is
another Industrial chemical that Is used In the manufacture of various dyes.
The compound is a carcinogen, and a large number of workers In the dye
Industry are exposed. Other arylamlnes in the dye industry may also prove
useful, such as benzidine or 2-napthylamine. Since benzidine is a metabolite
of most benzidlne-type dyes, benzidine-DNA adducts may indicate exposure to
benzidlne-type dyes. Exposure to the arylamine 4-aminobiphenyl has been
measured (199, 212), so this comound should also be given consideration.
Finally, psoralens might also prove useful as test compounds. These compounds
are used therapeutically for a variety of skin diseases. If adducts are found
In the blood or urine after treatment, It may be that an exposure-dose
correlation can be found. At present, the EPA Is conducting a exposure-dose
feasibility study with the compound Psoraben in a collaborative study with Or.
Reglna Santelll of Columbus University. They are looking at the dose-response
of adducts formed In hemoglobin and VBC DNA.
In addition, several compounds have been Identified that are on the EPA
priority list and that may form adducts. Although no literature has been
located on these compounds, it may be useful to carry out some preliminary
experiments to determine If these compounds form adducts.
The compounds are:
1) Dlchloromethane
2) Carbon tetrachloride
51
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3) Polychlorinated biphenyls
4) Chloroform
5) Toluene
6) Formaldehyde
An Initial study with these compounds might Include a simple feeding
study to determine If the compounds do form adducts. If so, several log dose-
response experiments should be conducted to check linearity with Hb, serum
albumin, and DNA.
This is not meant to be an all Inclusive list as much as it is meant to
comprise suggestions for compounds^o be considered for environmental
monitoring. Both Immunoassay and P-postlabeling techniques should be
adaptable to nearly any DNA adduct that is characterized. Pro|gin adducts can
only be monitored by using immunoassay techniques because the P-postlabellng
technique is specific for DNA adducts. However, protein adducts should occur
at higher concentrations than the DNA adducts because of a lack of protein
adduct repair systems and because greater amounts of sample are available.
Therefore, sensitivity should not be an insurmountable problem.
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APPENDIX
SUMMARY OF DNA ADDUCT LITERATURE
The appendix summarizes the characteristics of DNA adducts that have been
published in the literature. The information is derived primarily from
literature from 1981 to the present, although some earlier literature is
included.
The table is organized as follows. The compounds are in the order
discussed in the text with simple alkylating agents listed last, again in
alphabetical order.
The Appendix is self explanatory except for the DNA adduct persistence
data. The data is presented in two ways: either as half-life data, or as
percent increase (XI) or percent decrease (%D) over a time period (H-hour, D-
day).
Other abbreviations are as follows:
— - Information not given in literature
N/A - Not applicable
If a section is left blank, it Indicates that the literature was not
available and that that particular Information was not given in the citation
abstract.
53
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COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
HEASUREHENT T
1-Napthylamine (1-NA)
N-Hvdroxv-l-NA
N-(deoxyquanosin-06-y1)-1-NA
Radioisotope
1-Napthvlamine (1-NA)
N-Hvdroxv-l-NA
2-(deoxyquanos i n-O*-vl)-1-NA
Radioisotope
1-Napthylamine (1-NA)
N-Hvdroxv-l-NA
N-(deoxyquanos in-06-vl)-1-NA
Radioisotope
1-Napthylamine (1-NA)
N-Hvdroxv-l-NA
2-(deoxvquanosin-06-vl)-l-NA and
MS
N-(deoxyquanosin-0*-yl)-l-NA
2-Napthylamine (2-NA)
N-Hvdroxv-2-NA
Imidazole rinq-oDened derivative of N-(deoxvauanosin-8-vl)-2-NA
Radioisotope
2-Napthvlamine (2-NA)
N-Hydroxv-2-NA
1-(deoxyquanos i n -N a-v 1)-2-NA
Radioisotope
2-Napthvlamine (2-NA)
N-Hydroxv-2-NA
l-(deoxvadenosin-N®-yl)-2-NA
Radioisotope
2-Napthvlamine (2-NA)
N-Hvdroxy-2-NA
Imidazole ring-opened derivative of N-(deoxyquanosin-8-yl)-2-NA
Radioisotope
2-Naothyl amine (2-NA)
N-Hydroxy-2-NA
Imidazole rinq-opened derivative of N-(deoxyquanosin-8-yl)-2-NA
Radioisotope
2-NaDthvlamine (2-NA)
N-Hydroxy-2-NA
1 -(deoxyquanos i n -N a -v 1)-2-NA
Radioisotope
2-NaDthylamine (2-NA)
N-Hydroxv-2-NA
l-(deoxyquanosin-Na-yl)-2-NA
Radioisotope
2-Naothyl amine (2-NA)
N-Hydroxv-2-NA
1-(deoxvadenos i n-N6-vl)-2-NA
Radioisotope
2-Napthylamine (2-NA)
N-Hvdroxv-2-NA
l-(deoxyadenosin-N6-yl)-2-NA
Radioisotope
2-Naothylamine (2-NA)
N-Hydroxy-2-NA
Mdeoxyquanos i n-N2-vl) -2-NA. 1 -(deoxvadenos i n-
MS
N6-yl)-2-NA and a rinq-opened deoxyquanosine adduct
54
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DOSE/RESPONSE
PERSISTENCE
SPECIES
TISSUES
REF
N/A
N/A
In vitro
N/A
104
N/A
N/A
In vitro
N/A
104
N/A
30% D 01—07
Rats
Ini. Site
60
N/A
In vitro
104
N/A
30% 0 Dl—D7
In vitro
N/A
105
N/A
30% 0 Dl 07
In vitro
N/A
105
N/A
30% 0 Dl D7
In vitro
N/A
105
60uno1/kq(A): ~2 adducts/10® bases
100% D 02—07
Ooq
Liver
33
60umol/kq(A): ~10 adducts/108 bases
10% D D2—07
Doq
Bladder
33
60umo1/kq(A):0
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTUflE
HCASURtHlNI If II
4-Acetvl aminobiphenyl (4-AABP)
H-llydroxv-AABP
3-(deoxyquanosin-N,-yl)-AABP
Radioisotope
4-Acetylamlnobiphenyl (4-AABP)
N-llvdroxv-AABP
N-(deoxyquanosin-8-yl)-AABP
Radioi solope
4-Acetvlaminobiphenv) (4-AABP)
H-Hydroxy-AABP
N-fdeoxyquanos in-8-v1)-4-ami nob i pheny1
Radio!sotope
4-Aminobiphenvl (ABP)
N-Hvdroxv-ABP
N-(deoxvquanosin-8-vl)-ABP
Radioi sotope
4-Aminobiphenvl (ABP)
N-llydroxv-ABP
N-(deoxvquanosin-Na-v1)-ABP
Radioisotope
4-Aminobiphenvl (ABP)
M-llydroxY-ABP
N-(deoxvadenosin-8-vl)-ABP
Radioisotope
4-Aminobiphenyl (ABP)
M-llydroxv-ABP
N-(deoxvquanos i n-B-y1)-ABP
Radioisotope
4-Aminobiphenyl (ABP)
N-llydroxy-ABP
N-(deoxyquanos i n-N'-yl)-ABP
Radioisotope
4-AminobIohenvl (ABP)
N-Jlydroxv-ABP
H-(deoxyadenosin B-yl)-ABP
Radioisotope
4-Afnlnobiphenyl (ABP)
H-llydroxy-ABP
N-(deoxyquanosin-B-yl)-ABP
Radioi sotope
4-/WninobiDhenvt (ABP)
N-llvdroxv-ABP
H-(deoxvquanosin-N3-yl)-ABP
Radioisotope
4-Affllnobiphenyl (ABP)
N-llydroxy-ABP
N-(deoxvauanosin-8-vl)-ABP
Radioisotope
4-Ace tv1ami no-4' - f 1uori biphenyl
N-Mydroxy-AAFBP
M-(deoxvadenos i n-8-y1)-AAFBP
4-Acetylam}no-4'-fluoribiphenyl
N-llydroxy-AAfBP
3-(deoxvquanosin-N2-yl)-AAFBP
3.2'-DimethYl-4-aminobiphenYl (DHABP)
N-llydroxy-DMABP
H-(deoxyquanosin-8-y1-)-DHABP
Radioi sotope
3.2'-DimeIhv1-4-ami nob i phenyl (OHABP)
N-llydroxy-DHABP
5-(deoxvquanos i n-M 2-y1)-DHABP
Radioisotope
56
-------
DOSE/RESPONSE
PERSISTENCE
SPECIES
TISSUES
REF
4&nq/kq(A): 2.4 fmol adduct/uq DNA
62% D DO.02-01: 62% D 01-029
Rat
Liver
75
40mq/kq(A): 1.5 fmol adduct uQ DNA
80% D 00.02-01: 100% D 01-09
Rat
Liver
75
40mq/kq(A): 13.2 fmol adduct uq DMA
77% D DO.02-01: 93% D D1-029
Rat
Liver
75
N/A
N/A
In vitro
N/A
33
N/A
N/A
In vitro
N/A
33
N/A
N/A
In vitro
N/A
33
60unol/ka: ~ 300-800 adducts/108 nucleotides
~ 130% I D1-D7
Doq
Liver
33
60unol/ka: ~ 0
-------
COMPOUND
ACTIVE METABOLITES
APDUCT STRUCTURE
MEASUREMENT TECH
1",M" —J'
3.2*-Dimethyl-4-ami nob iDhenvl
(DMABP)
N-Hvdroxv-OMABP
5-(deoxyquanosin-N*-vl)-DMABP
Radioisotope
3.2'-0imethv1-4-aminobipheny1
(DHABP)
N-hydro*y-OHABP
5-(deoxyquanosin-Na-vl) DMABP. N-(deoxyquanosin-
8-y1)-0MABP
¦ ¦
MM
nu
2-Acetvlaminofluorene (AAF)
M-Hvdroxv-AAF
N-(deoxyquanosin-8-vl)-AAF
2-Acetyl aminofluorene (AAF)
M-Hydroxy-AAF
3-(deoxyquanosin-Na-8-vl)-AAF
2-Acetvlami nofluorene (AAF)
M-Hvdroxv-AAF
N-(deoxvquanosin-8-y1)-AAF
2-Acetvlaminofluorene (AAF)
N-Hvdroxv-AAF
N-(deoxyquanosin-8-vl)-AAF
Radioisotope
2-Acety1ami oof1uorene (AAF)
M-Hvdroxv-AAF
3-(deoxyquanosin-8-yl)-AAF
Radioisotope
2-Acetvlaminofluorene (AAF)
N-Hvdroxv-AAF
N-(deoxvQuanosin-8-vl)-AF
Radioisotope
2-Acetylaminofluorene (AAF)
N-Hvdroxv-AAF
N-(deoxvquanos i n-8-vl)-AF
Radioisotope
2-Acety1aminofluorene (AAF)
M-Hvdroxv-AAF
N-(deoxyquanosin-8-v1)-AF
Radioisotope
2-Acety1 ami nofluorene (AAF)
N-Hvdroxv-AAF
N-(deoxvquanosin-8-vl)-AF
Radioisotope
2-Acetylaminofluorene (AAF)
M-Hvdroxv-AAF
(Guan-8-vl)-0NA Adducts
Immunoassay
2-Acety1 ami nofluorene (AAF)
N-Hvdroxv-AAF
(Guan-8-yl)-DNA Adducts
Inmunoassay
58
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
0.5 irmol/ko; ~ 40 pmol DHABP/mo DMA (est.) ~ 7OX D D1-D7 Rat Internal mucosa 229
0.5 innol/kq; ~ 10 pmol DMABP/mq DNA (est.) ~ 70% D PI-07 Rat Internal mucosa 229
7OX D 00-07 Rat Intestine 230
29X 0 00-07
Rat
Intestine
230
T 1/2 = 7D
Rat
Liver
123.124
Rat
Liver
123.124
Rat
Liver
29
10 mq/kq: biweekly to 6 weeks
1-1.8 pmol AAF/mq DNA
100X D 01-014
Rat (male)
Liver
31
10 mq/kq: biweekly to 6 weeks
0.8-3.2 pmol AAF/mq DNA
20-90% 0 01-014
Rat (male)
Liver
31
10 mq/kq: biweekly to 6 weeks
12-58 pmol AAF/mq DNA
10-50% 0 Dl-014
Rat (female)
Liver
31
10 mq/kq: biweekly to 6 weeks
15-20 pmol AAF/mq DNA
50-80% 0 01-014
Rat (male)
Liver
31-
10 mo/kq: biweekly to 6 weeks
5-16 pmol AAF/mq DNA
10% 1-60% 0 Dl-014
Rat (female)
Kidney
31
10 mo/kq: biweekly to 6 weeks
2-4 pmol AAF/mq DNA
~ 50-80% D Dl-014
Rat (male)
Kidney
31
0.02% in feed, up to 60 days:
80-238 fmol adduct/uq ONA
50% 0 01-028
Rat (male)
Liver
175
0.04X in feed, up to 46 days; 129-252 fuel adduct/uq DNA 9OX D D1-028 Rat (male) Liver 175
59
-------
COMPOUND
ACTIVE METABOLITES
ADOUCT STRUCTURE
MEASUREMENT T
2-Acetyl aminofluorene (AAF)
M-Hydroxy-AAF
(Guan-8-yl)-DNA Adducts
Irmunoassay
2-Acetylami nofluorene (AAF)
M-Hydroxy-AAF
(Guan-8-yl)-DNA Adducts
Itranunoassay
2-Acetylaminofluorene (AAF)
N-Hvdroxy-AAF
(Guan-8-v1)-DNA Adducts
Immunoassay
2-Acetylaminofluorene (AAF)
M-Hydroxy-AAF
N-(deoxyquanosin-8-y1)-AF
Radioisotope
2-Acetylaminofluorene (AAF)
M-Hydroxy-AAF
N-(deoxyquanosin-8-vl)-AF
Radioisotope
2-Acetylaminofluorene (AAF)
M-Hydroxy-AAF
N-(deoxyquanos i n-8-vl)-AF
Radioisotope
2-Acetylaminofluorene (AAF)
N-Hvdroxy-AAF
N-(deoxyquanosin-8-yl)-AF
Radioisotope
Mia
M U
M U
Hit
MM
M-Hydroxy-AAF and N-Acetoxy AAF
"" and 3-(deoxyquanosin-N2-yl)-2-AAF
Radioisotope
MM
M-Hydroxv-AAF
N-(deoxyquanosin-8-vlJ-2-AAF
MM
MM
UN
"" and 3-(deoxyquanosin-Na-yl)-AAF and
UM
AAF-N-7 adducts with quanosine
Benzidine (BZ)
M-Hydroxy-U'-acetyl-BZ
N-(deoxyquanos i n-8-yl)-N'-acetyl-BZ
Radioisotope
Acetyl-BZ
N-Hydroxy-N'-acetyl-BZ
M-(deoxyquanos in-8-y1)-N'-acetyl-BZ
Radioisotope
Acetyl-BZ
N-Hydroxy-N'-acetyl-82
N-(deoxyquanosin-8-yl)-N'-acetyl-82
Radioisotope
Diacetyl-BZ
M-Hydroxy-N.N'-diacetv1-BZ
N-(deoxyquanos i n-8-y1)-N.N'-d iacetyl-BZ
Radioisotope
60
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
0.02X In feed, up to 60 days; 14-31 fmol adduct/uo DMA 45X D D1-028 Rat (male) Kidney 175
0.04X in feed, up to 60 days: 15-47 fmol adduct/uo DMA SOX D D1-028 Rat (male) Kidney 175
0.04X in feed, up to 60 days; 0-27 fmol adduct/uq DMA 65X D 01-028 Rat (male) Adrenal 175
60 umol/kq; 500 adducts/108 nucleotides 80% D 02-07 Dog Liver 33
60 umol/kq: 100 adducts/108 nucleotides 70% D D2-P7 Dog Bladder 33
House Liver 127
= T 1/2 = 6D Rat Liver 124. 228
40 mo/kg; 1.5 adducts/10* nucleotides 5 1/2 = 14.20 Rat Mammary gland 6
— N/A In vitro N/A 211
1-230 uH: - — Salmonella typhimurium N/A 37
— N/A In vitro N/A 214
80 pan in water for 1 week: ~ 90 pmol BZ/mq DNA SOX D D0-D1; 60X D DO-07 Rat Liver 138
ill unol/kq; 70 pmol/mq OHA 60X 0 01-07 Rat Liver 229
111 tfipl/ko: 33 ptnol/mo ONA 60X D 01-07 Hamster Liver 229
111 umol/kq; 5 pmol/mq DNA Rat Liver 229
61
-------
COMPOUND
ACTIVE NETABOLITES
ADOUCT STRUCTURE
MEASUREMENT 1
N-Nethvl-4-aminoazobenzene (NAB)
N-Sulfonvloxv-NAB
N-(deoxyquanosin-8-vl)-NAB
Radioisotope
N-Nethvl-4-aminoazobenzene (NAB)
N-Sulfonvloxy-MAB
3-(deoxyquanosin-N2-vl)-NAB
Radioisotope
N-Nethvl-4-aminoazobenzene (NAB)
N-Su1fonvloxy-NAB
3-(deoxvadenosin-N6-yl)-NAB
Radioisotope
N-Nethvl-4-aminoazobenzene (NAB)
N-Sulfonvloxv-NAB
N-(deoxvquanosin-8-vl)-NAB
Radioisotope
N-Nethvl-4-aminoazobenzen (NAB)
N-Sulfonvloxv-NAB
N-(deoxvquanosin-B-yl)-NAB. plus N-7 substituted adducts
Radioisotope
with quanine
me
M-sulfonyloxy-NAB
3-(deoxvquanosinNa-vl)-NAB
Radioisotope
4-Ami noazobenzene
N-Hvdroxy-AB (?)
N-(deoxyquanosin-8-vl)-A8
Radioisotope
2-Acetyl aminoohenanthrene (AAP)
N-Hvdroxv-AAP
N-(deoxyquanosin-8-ul)aminophenanthrene (AP)
S2P-Postlabelinq
Benzo(a)pvrene (BaP)
(+)7.8-hvdroxv-9.10-epoxy BaP (BPOEI)
Radioisotope
BaP
(-)7.8-hvdroxv-9.10-eDOxv BaP (BPDEII)
Radioisotope
BaP
BPOEI. BPDEII
N7 adduct with quanine: adenine adducts. N-2 quanine add.
Radioisotope
BaP
BPDEI. BPDEII
Radioisotope
BaP
BPOEI. BPDEII
BBDEI-ouanosine. BPOEI-protein
Radioisotope
BaP
Radioisotope
BaP
BPOEI. BPDEII
Radioisotope
62
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
0.2 mnol/kq; days 1.3.5.B 0.1-2 adducts/10* nucleotides Rat Liver 216
0.2 mnol/kq. days 1.3.5.8 0.1-1 adducts/10* nucleotides Rat Liver 216
0.2 mnol/kq. days 1.3.5.8 0-0.9 adducts/106 nucleotides Rat Liver 216
120 mg/kq; ~* 8 umol adduct/mol ONA T 1/2 ~ 50 House Liver 213
— N/A In vitro N/A 214
120 mq/ko ~ 1.5 umol adduct/mol ONA
T 1/2 between 5 and 10 D
House
Liver
213
House (pre-weanlinq)
Liver
54
40 ma/ko: 27 fmol adduct/uo ONA
50% 01-09
Rat
Liver
75
Hunan
Colon explants
39
Human
Colon explants
39
Numerous
Numerous
14.
203
Linear 0.01-300 uq/mouse
House
Epidermis
163
Linear 2-11 umol/kq siqmoidal 11-135 umol/kq
House
Lunq. liver, forestomach
3
1 unol/ko — 10 umol/ka — 3-5 X increase
Rat
Lunq. liver
39
incubated luH BaP 10-30X adduct level diference
Human, various others
Bladder, trachea
51
63
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
MEASUREMENT TECH
BaP
BPDEI. BPDEII
Protein
Fluorescence
BaP
BPOEI. BPDEII
Radioisotope
BaP
BPDEI. BPDEII
BPDE-dG. BDDE-dA
Radioisotope
BaP
BPDEI. BPDEII
BPDE-dG. BDDE-dA
Radioisotope
BaP
BDEI
BDDEI-ONA
Radioisotope
BaP
BDEI
BDDEI-ONA
Radioisotope
Benzo(a)ovrene
BPOE
Protein adduct (Hemoqlobin)
Fluorescence
MM
BPDE-I
7R BPDE I-deoxvauanosine
Radioisotope
mm
UN
BPDE-N3-deoxvquanosine and
9-hydroxybenzo(a)pvrene-DNA
Radioisotope
mm
BV
MM
MM
mu
a m
7R. 7S BPDE-N2-deoxvquanosine
Radioisotope
mm
a «
MU
MM
mm
MM
MM
Mil
mm
an
MM
MM
64
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
Linear 50-500 yig/mouse T 1/2 = ~2D. T 1/2 = ~ 30D House Skin 195
Linear 50-500 tig/mouse House Hemoglobin 195
200Bmol/mouse —7—15 pmol BPOE/mg macromolecule T 1/2 ~ 2-3D (all) House Skin. DMA. RMA. protein 162
2SQwno1/mouse 5.2 pmol BPDE/mq DMA 76% D Dl-07 93% D D1-0-21 House Skjn 5
IQOOmol/mouse 12.36 pmol BPDE/mq ONA 56% D Dl-07. 100% D D1-D21 Rat Skin 5
6 mq BaP/mouse 6. 7 pmol BPDEI/mQ DMA (Lung. Liver) T 1/2 (lung 20P. liver 140) A/HeJ mouse Lung, liver 125
6 mq BaP/mouse 6. 3 pmol BPOEl/mg DMA (lunq. liver) T 1/2 (lung 15D. liver 3D) C57BL/6J mouse Lung, liver 125
T 1/2 = 6D House Hemoglobin 194
95% D 00-021 House Skin 4
200 nmol/mouse; 3.3 pmoles/mq DNA — Swiss mouse Skin LI
200 rmol/rat: 0.74 pmoles/mg DMA 33% 0 H12-H48 Rat Skin 12
1.5 uH BP0E1; 15 umol/mol DMA 56% D H0=H8 67% 0 H0-H24 Human Fibroblasts !H
—; 14.2 pmol/mo DMA 40% 0 D1-P3 (total adducts) Rat Enfcryo cells 23
—: 6.0 pmol/mg DMA 47% D D1-P3 (total adducts) Hamster Embryo cells 23
—: 4.0 pmol/mg DMA 30% D Dl-03 (total adducts) Human Hepatoma cells 23
65
-------
COMPOUND
ACTIVE METABOLITES
ADOUCT STRUCTURE
MEASUREMENT TECH
• a
ua
9-OH-8P-4.5-epoxided-dG. 7S. 7R-BPDEL-dG. and
7R-8PDE II-dG
Radioisotope
mm
UN
Trans-7 R-6PDEI -N 2-deoxyquanos i ne
Imnunoassav
10-Azabenzo(a)pyrene-4.5-ox ide
Direct actinq
9-Anthroyloxirane (9-AO)
Direct actinq
N-3 adduct of 9-AO to adenine
UV-absorfoance
1-Oxiranylpyrene
Direct actinq
Similar to BaP (?)
UV-absorbance
3-Methylcholanthrene (3-MC)
3-MC Diolepoxide
3 MC
3-MC Diolepoxide
Radioisotope
3 MC
3-MC Diolepoxide
Radioisotope
3 MC
3-MC Diolepoxide
Radioisotope
3 MC
3-MC Diolepoxide
Radioisotope
3 MC
3-MC Diolepoxide
Radioisotope
3 MC
3-MC Diolepoxide
Radioisotope
3 MC
3-MC Diolepoxide
Radioisotope
3 MC
3-MC Diolepoxide
Radioisotope
66
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
4 doses 1.2 mnol/mouse; 1 adduct 6.2 x 10* cells 1 D < T 1/2 < 6D Mouse Skin 178
5.5 uH BaP; (0.75. 0,33. 3.0. 1.34) » 10T moles 61-73X 0 DO-02 Human Fibroblasts HO
Mducts/mol OKA
Nonlinear 50-1500 wnol/ 2.3-11 fmol uq DNA T 1/2 = 3D House Skin 154
157
M/A In vitro M/A 237
M/A In vitro M/A 117
== Human Bronchus, colon, esophagus 77
31 wnol 3-HC 45 fmol adduct/mq DMA 62* 0 DO-D28 A/J mouse Lung 203
31 wnol 3-HC 5.S fmol adduct/mq DMA 100% P DO-028 A/J mouse Liver 203
31 wnol 3-HC IB fmol adduct/mq DMA 38* D 00-028 C3H/HeJ mouse Lung 203
31 nmol 3-HC 4.4 fmol adduct/mq OMA 100% D 00-028 C3H/HeJ mouse Liver 203
31 wnol 3-HC 16 fmol adduct/mq OMA 81X D DQ-028 DBA/2 J mouse Lung 203
31 nmol 3-HC 4.8 fmol adduct/mg DMA 100K D DO-028 0BA/2J mouse Liver 203
31 wnol 3-HC 16 fmol adduct/mq OMA 71% D DO-028 C57BL/6J mouse Lung 203
31 wnol 3-HC 9.7 fmol adduct/mq DMA 100% 0 DQ-028 C57BL/6J mouse Liver 203
67
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
HEASUREHENT '
7. 12-Oimethylbenzra]anthracene (DHBA)
DHBA 3.4-diol-l,2-epoxide.
DBHA-ONA
Radioisotope
7-hydroxymethyl-12-methy1benz
(a)anthracene diol-epoxide
7.12-Dimethvlbenzralanthracene (DHBA)
U
DBHA-ONA
Radioisotope
7.12-Oimethylbenzralanthracene (DHBA)
U
DBHA-ONA
Radioisotope
7.12-Dimethy1benz[a]anthracene (DfffiA)
u
DBHA-ONA
Radioisotope
7.12-0imethv1benz[a]anthracene (DHBA)
Svn-OMBA-diol-epoxide
Syn-OHBA diol-epoxide-dAdo
Radioisotope
7.12-Oimethvlbenz[a]anthracene (DfffiA)
Anti DHBA diol-epoxide
Anti DBHA diol-epoxide-dAdo. dGuo
Radioisotope
15.16-Dihydro-l1-methylcyclopenta-
1.2-epoxy-3.4-di hydroxy-11-methyl ketone
11-methyl ketone diol-epoxide-ONA
Radioisotope
fa]phenanthrene-17-one(11-methylketone)
11-methylketone
1.2-epoxy-3.4-d i hydroxy-11-methyl ketone
11-methyl ketone diol-epoxide-ONA
Radioisotope
11-methvlketone
1.2-epoxv-3.4-dihydroxy-11 -methyl ketone
11-methvl ketone diol-epoxide-DNA
Radioisotope
11-methylketone
1.2-epoxy-3.4-dihydroxy-11-methyl ketone
N2 quanine adduct with 11-methylketone diol-epoxide Absorbance MS
11-methylketone
1.2-epoxv-3.4-dihydroxy-11-methyl ketone
Radioisotope
11-methylketone
1.2-eDOxy-3.4-dihydroxv-11-methyl ketone
Radioisotope
7-8romomethvl benzanthracene (7-BffflA)
Radioisotope
68
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
20. umol/kg: 69 umol DHBA/mol DNA 73% D D2-014 Long-Evans rat Liver 50
20 unol/ko; 53.3 umol/DMSA/mol QUA 31% D D2-014 Long-Evans rat Hamnary gland 50
20 umol/ko: 37.8 umol OHBA/mol DNA
26% D 02-014
Soraoue-Dawlev rat
Liver
50
20 umol/ka: 28.6 umol DMBA/mo 1 DNA
1% 0 D7-014
Spraoue-Oawley rat
Mamnary gland
50
0.01 imiol/mouse — 0.1 umol/mouse — 3-4X1
11-25* D D1-D2
NIH-Swiss. C55BL mice
Skin
59
0.01 umol/mouse — 0.1 umol/mouse — 3-4X1
2-34% 0 01-02
NIH-Swiss. C55BL mice
Skin
59
N/A
In Vitro
N/A
1. 203
House
Skin
1. 203
House
Embryo cells
203
N/A
In Vitro
N/A
231
3 mo/mouse: 283. 345 nmol adduct/mol DNA
T 1/2 ~ 6.5 0
House
Skin, luna
2
3 mq/mouse. 641 nmol adduct/mol DNA
T 1/2 ~ 2.5 0
House
Liver
24
7 nmol/mouse. 44 adducts/107 nucleotides
T 1/2 < 1 0
House
Liver
24
69
-------
COMPOUND
ACTIVE METABOLITES
AOPUCT STRUCTURE
MEASUREMENT TECH
7-flrcmomethyl benzanthracene (7-BH8A)
Radioisotope
7-firomomethvl benzanthracene (7-BMBA)
Radioisotope
1-Nitroovrene (1-NP)
N-hvdroxv-1-aminoDvrene
N-(deoxvquanosi n-8-vl)-1-ami noDvrene
Radioisotope
l-Nitroovrene (1-NP)
N-hvdroxv-1-ami noDvrene
Radioisotope
1-Nitropyrene (1-NP)
N-hvdroxv-1-ami noovrene
N-(deoxvquanosi n-8-vl)-1-ami nopyrene
Radioisotope
1.8-dinitropvrene
1 -ami no-8-ni troDvrene
N-(deoxvquanosin-8-vl)-l-amino-8-nitropvrene
Radioisotope
Dibenzola.e)fluoranthene (DBF)
3.4-dihvdroxv-1.2-epoxv-OBF
N3-adduct with quanine
MS
Dibenzo(a.e)fluoranthene (DBF)
12.13-dihvdroxv-l 1.2-ei>oxv-OBF
N3-adduct with auanine
MS
Dibenzo(a.e)f1uoranthene (DBF)
12.13-dihvdroxv-ll.2-epoxv-OBF
Radioisotope
S-methylchrysene (5-MC)
1.2-dihvdroxv-3.4-ei>oxv-5MC (DEII) and
Na-quanosine adducts with OE-I and DE-1I
Radioisotope
7.8-dihvdroxv-9.10-eooxv-5HC (DEII)
5-MC
OEI. DEII
Radioisotope
4—Nitroaui noline-1-ox ide
O-acetyl.O.O'-diacetvl-
N-(deoxvquanosin-8-yl)-4N00
(4N00)
4-hvdroxvami noau i noli ne
4-Nitroauinoline-l-oxide
O-acetvl.O.O'-diacetvl-
N-(deoxyquanosin-8-v1)-4N00
Radioisotope
(4NQ0)
4-hydroxvami noau i noli ne
70
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
7 nmol/mouse. 48 adducts/107 molecules T 1/2 ~ 15 D House Liver hi stone 24
7 nmol/mouse. 1400 adducts/10' molecules T 1/2 ~ 1 D mouse Liver albumin 24
— Salmonella typhimurium N/A 87. 96
100 uHl-MP. 103 pnol 1-NP/mg DMA N/A In Vitro N/A 25
8.1 uH 1-1NP; = Rabbit Lung 98
3 uH l-amino-8-nitropyrene; — Salmonella typhimurimi 96. 87
— N/A In Vitro N/A 166
In Vitro N/A 166
SOX 0 DO-02 House enfcryo Fibroblasts 167
— In Vitro N/A 144
144
70 nwol/mouse. ~ 3 cmol adducts/mo ONA 20X D DO-02 House Skin 142. 143
-- In vitro N/A 21. 67
unclear Rat Pancreas, liver, kidney, lung blood 56
71
-------
COMPOUND
ACTIVE METABOLITES
AOOUCT STRUCTURE
MEASUREMENT TECH
w 1 I"' "1 .... " « ' - w. ¦ -* ¦ - 1" * — ^
0xo-Ns-pvrimidyl)-9-hvdroxy AFB^, and 8.9-
Dihvdro-8-(N7-quanvl)-9-hvdroxv AFB.
Aflatoxin B. (AFB)
n a
Radioisotope
Aflatoxin B. (AFB)
AFB -2.3-epoxide
2.3-dihYdro-2-(Ns-formvl)-2'.5'.6'-triamino-4'-
Radioisotope
Oxo-N'-ovrimidvD-S-hvdroxvaflatoxinBj. and
2.3-d ihvdro-2-(N 7-quanvl)-3-hvdroxvaf1atox i n B,
Aflatoxin B. (AFB)
AFB -2.3-epoxide
MM
Aflatoxin B. (AFB)
AFB -2.3-eix>xide
U II
Radioisotope
Aflatoxin B. (AFB)
AFB -2.3-epoxide
2.3-d ihydro-2-(N7-quanv1)-3-hvdroxvaf1atox in Bt
Absorbance
Steriamatocvstin (STO)
ST-1.2-epoxide
ST-M-quanine adduct
saDOSt-labelinq
Mitmycin C (MHC)
WIC-reduction product
Na-qua-.06-qua-. and N6-Ado-MMC
Absorbance
3-amino-l-methvl-5H-ovridor4.3-bl N-OH-Tpo-P-2
3-(C 8-quanyl)ami no-1 -methv1-5H-pyr i do[4.3-b ]
Absorbance
Indole (Gua-Trp-P-2)
2-ami no-6 -methyl -d i Dvri do
M-OH-Glu-P-1
2-(C8-ouanvl)amino-60n)ethYldipvrido^.2-a:3,.
Absorbance
n.2-a:3*.2'-d]imidazo1e (Glu-P-1)
2'-dlimidazole (Gua-Glu-P-1)
72
-------
POSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
I uH AFB. 2.2 — 135 u mol AFB/mol DMA == Human, rat, dog, hamster Trachea, or bronchial tissues 202
1 wH AFB. 1.5 — 26 umol AFB/mol DMA — ^ Bladder tissues 202
0.3 uH. 5-8 umol adduct/mol DMA Tl/2 ~ 12 H House Embryo fibroblasts 8
— N/A In vitro N/A 4?
0.6 mq/kg; 268 onol/mg DMA T 1/2 (> 72H. 7.5H) Rat Liver 48
Linear 0.125 — 0.5 mq/kq; siqmoida 10.125 10 mq/kq N/A Rat Urine 35
Linear 1-9 mq/kq . 1 oqarhvthmic 0.33-9 mq/kq T 1/2 12 h. 70. 1090 Rat Liver 1B3
N/A In vitro N/A 84
— N/A In vitro N/A 82. 85
N/A In vitro N/A 82. 85
73
-------
COMPOUND
ACTIVE HETABOLITES
AOOUCT STRUCTURE
MEASUREMENT TECH
3-amino-4.6-dimethvlpvridori.2-a: H-AcO-AGlu-P-5 Clu-P-3 bound to Ca of guanine
3' .2-d]imidazole(aGlu-P-3)
• ¦ - —
Safrole
1'-Hvdroxvsa fro1e-der i vat i ons
Na(trans-isosafrol-3'-vl)deoxvauanosine and
Radioisotooe
M6-(trans-isosafrol-3'-vlIdeoxvadenos in
Safrole
1 '-Hvdroxvsafrole-derivations
•III
Radioisotope
Safrole
1'-Hydroxvsafrole-derivations
HU
Radioisotope
Safrole
1'Hydroxvsafrole-derivations
The 2 adducts above plus 8-(trans-isosafrol-3'-yl)-
Radioisotope
and 7-(trans-isosafrol-3'-vl)quanine
Estraqole
1'-Hydroxyestraqole-derivations
N2-(estraqol-l'-vl)deoxyquanosin.N3-(cis-
Radioisotope
isoestraqol-3'-vl)deoxvauanos i ne. trans-i soest raqol-
3'-vl)deoxyquanosine. and N6-(trans-isoestraqol-
3'-v1)deoxvadenos i ne
Estraqole
1'-Hydroxyestraqole-derivations
IIII
Radioisotope
Estraqole
1'-Hvdroxvestraqole-derivat ions
MM
Radioisotope
Estraqole
>aP-post labelinq
Allylbenzene
MM
74
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
136
12 unol/mouse 350 pmol/mg DMA T 1/2 ~ 2D House Liver 170
12 unol/mouse. 275 cmol/mq tRHA T 1/2 ~ 2D House Liver tRNA 170
12 unol/mouse. 250 pnol/mq protein T 1/2 ~ 2D House Liver protein 170
— — House Liver 234
12 unol/mouse. 250 pmol adduct/mq DMA T 1/2 ~ 4D (total adducts) House Liver 169
12 unol/mouse; 200 pmol adduct/mq tRMA T 1/2 ~ 2.5D (total adducts) House Liver tRNA 169
12 unol/mouse; 250 pmol adduct/mq protein T 1/2 ~ 2D (total adducts) House Liver protein 169
10 ma/mouse; ~ 290 pmol adduct/mq DNA Persistent after 43 D House Liver protein 171. 179
10 mq/mouse; 3.1 pmol adduct/mq DMA ™ House Liver protein 171, 179
75
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
MEASUREMENT TECH
Safrole
Hvristicin
•aP-postlabelinq
Dili aoiol
_ N M
Parslev aoiol
_ NM
Isosafrole
MM
Methyleuqenol
H M
Elemicin
« M
Anethole
m m
TrimethvlDsoralen
UV activated
Linked to 5.6 positions of thymine (cvcloadduct)
8-methoxvosora 1 en
UV activated
UN
Anqelicin
UV activated
on _
5-methvli sopsora1en
UV activated
NN __
Monocrotaline (MC)
Pyrrole form of MC
7 position of MC bound to HJ position of deoxyquanosine
CC-1065
bound to Ns position of adenine
Cis-diamminedichloroDlatinum (II)
Direct actinq
Intra, interstrand links to N7 of quanine: DNA protein crosslinks Iirmunoassay
nu
AA
76
-------
POSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
10 mq/mouse: 205 pmol adduct/mq DNA
Persistent after 43D
House
Liver. Drotein
171. 179
10 mq/mouse 50 pmol/mq DNA
Persistent after 43D
House
Liver
171. 179
10 ma/mouse 40 pmol/mq DNA
Persistent after 43D
House
Liver
171. 179
10 ma/mouse 14 pmol/mq DNA
House
Liver
171. 179
10 mq/mouse 4.5 pmol/mq DNA
House
Liver
171. 179
10 mq/mouse 196 umol/mq DMA
Persistent after 43D
House
Liver
171. 179
10 mq/mouse 16 pmol/mq IMA
Persistent after 430
House
Liver
171. 179
10 mq/mouse 1.3 umol/mq DNA
House
Liver
171. 179
44
44
44
44
185
206
Linear. 0-600 mq/Ha. 0-200 attomol/uq DNA
Han
White blood cells
177
T 1/2 6 min-45D Various Various 185a
77
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
MEASUREMENT TECH
¦ • MM 1*11
Cis-diamninetetrachloroplatinum (IV)
IIU
M II
AA
Trans-di airmi ned 1 chl oropl at i um
-------
DOSE/RESPONSE
PERSISTENCE
SPECIES
TISSUES
REF
Linear 25-200 cim. 0.8-2.5 crosslinks/109 daltons
T 1/2 ~ 24 H
Chinese hamster
Cultured ovarv cells
173
MM
U t>
u u
uu
173
MM
UN
MM
MM
173
Linear 100-800 uH 0.7-2.2 crosslinks/10 *daltons
MM
MM
173
DES treatment for 8 months
Hamster
Kidney
133
Linear 0-0.7 off: 0-0.5% nucleosides adducted
In vitro: salmonella tvphimurin
N/A
55
Linear 0-0.08 nM: 0-3% nucleosides adducted
MM
N/A
55
Linear 0-0.03 irM: 0-40 renol PHZ/ma protein
M U
Protein
55
In vitro
201
Non-linear 1.1-4.9 nmol/ka: 17-31 rwnol/q DNA
House
Liver
41
Non-linear 1.1-4.9 mmol/kq: 3-60 nmol/g Hb
House
Liver
41
79
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
MEASUREMENT TECH
mm
an
UN
Stvrene 7.8 oxide
Direct actinq
Same as stvrene
Htt
• N
HU
HU
on
HH
Ethylene oxide
«i n
MH
Ethylene oxide
HU
«H
Stvrene 7.8 oxide
n u
U«
NN
no
« M
MM
»H
U M
MM
n u
Hvdroxvlamine
Direct actina
N4-Hvdroxvcvtos i ne
Dimethylcarbamyl chloride
Direct acting
6-d imethylcarbamy1oxy-2'-deoxyquanosi ne
MS
6-dimethylamin-2'-deoxvquanosine. and 4-
di methv1aminothvmidine
Dfethvlcarfcamvl chloride
Direct actinq
6-d i et hylcarbamy1oxy-2'-deoxyquanos i ne
MS
Trans-4-acetvl ami nosti1 bene
Radioisotope
80
-------
DOSE/RESPONSE
PERSISTENCE
SPECIES
TISSUES
REF
Non-liner 1.1-4.9 mnol/kq: 200-430 nmol/q protein
House
Liwer
41
1.1 mmol/kq: 8 nmol/q ONA
House
Liver
41
Non-linear 0.037-1.1 mnol/kq: 0.1-13 nmol/q Hb
House
Liver
41
Non-linear 0.037-1.1 mnol/kq 9.8-750 nmol/q protein
House
Liver
41
0.044 nmol/ko: 2 nmol/q DNA
House
Liver
41
0.044 nmol/ko 2 nmol/q Hb
House
Liver
41
0.36 mnol/kq: 5 nmol adducts/q ONA
House
Brain
41
0.36 mnol/kq: 3 nmol adducts/q DNA
House
Lunq
41
0.36 mnol/kq: 0.6 nmol adducts/q DNA
House
Spleen
41
0.36 mnol/kq: 0.3 nmol adducts/q DNA
House
Testis
41
100
_
191
191
S unoi/kq; twice weekly for 6 weeks; 24 pmol/mq DMA T 1/2 = 220 Rat Liver 93
81
-------
COMPOUND ACTIVE METABOLITES AODUCT STRUCTURE MEASUREMENT TECH
M a _ M w
¦ M MM
MM ^ tan
»• ^_ . MM
MM ___ MM
T rans-4-acetvl ami nos t i1bene
Trans-4-acetvl ami nos t i1bene
Trans-4-acetvl ami nost i1bene
Trans-4-acetvl ami nos t i1bene
l-bromo-2-chloroethane
S-(2-bromoethvl)GSH or S-[2-(N7-quanvl)ethvl]GSH
S-(2-bromoethyl)GSH
N-butvlmethanesulfonate
Direct actinq 06-n-butvl-quanosine. N7-n-butvlquanosine and Na-n-butvladenosine Radioisotope
N-n-butyl-N-ni trosourea
Direct actinq 06-n- and sec-butvlquanosine. N7-n- and sec-butylquanosine Radioisotope
and 3-n-butvladenosine
02
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REP
5 umol/kq; twice weekly for 6 weeks; 18 pmol/mq DMA Persistent for 6 weeks Rat Kidney 93
5 unol/kq; twice weekly for 6 weeks; 2 pmol/mq ONA T 1/2 a 18D Rat Lung 93
S umol/kq; twice weekly for 6 weeks: 3 pmol/mq QUA T 1/2 = 9.OP Rat Gland stomach 93
S unol/kq; twice weekly for 6 weeks; 27 pmol/kq protein T 1/2 = 5.10 Rat Liver, protein 93
5 unol/kq; twice weekly for 6 weeks; 10 pmol/kq protein T 1/2 = 8.70 Rat Kidney, protein 93
5 unol/kq; twice weekly for 6 weeks; 4 pmol/kq protein T 1/2 = 7.30 Rat Lung, protein 93
5 unol/kq; twice weekly for 6 weeks; 7 pmol/kq protein T 1/2 = 6.SO Rat Gland stomach, protein 93
S unol/kq; twice weekly for 6 weeks; 2 pmol/mq RHA T 1/2 = 4.5D Rat Liver 93
S unol/kq; twice weekly for 6 weeks; 16 pmol/kq RHA Persistent over 6 weeks Rat Kidney 93
S unol/kq; twice weekly for 6 weeks: 1 pmol/kq RHA T 1/2 = 20.OP Rat Lung 93
5 unol/kq; twice weekly for 6 weeks; 1 pmol/kq RHA T 1/2 s 5.00 Rat Gland stomach 93
2 mH; 217 pmol/mq OKA — In vitro N/A 160
Linear 1-10 n*: 3 lOrrrt 4.1. 188. 10.6 umol/mol PHA — In vitro N/A 187
Linear 1-10 nrt: § lOrrft 99. 29. 151. 17. and 155 umnol/PNA — In vitro N/A 187
83
-------
COMPOUND
ACTIVE METABOLITES
AOOUCT STRUCTURE
MEASUREMENT TECH
l-(2-ch1oroethy1)-3-(cis-2-hydroxy) Direct acting 7-hydroxyethyl aquanine and 7-chloroethyl guanine Radioisotope
cvclohexyl-1-nitrosourea
l-(2-ch1oroethyl)-3-cyclohexyl-
Direct acting
M II
RadioisotoDe
1-nitrosourea
1-chloroethyl(methyl sulfonyl)-
Direct acting
nn
Radioisotope
methanesulfonate
2-chloroethyl(methyl sulfonyl)-
Direct acting
7-chloroethylquanine
methanesulfonate
M2-chloroethvl)-l-nitrosourea
Direct acting
lilt
Radioisotope
1.2-0 i bromoethane
S-(2-bromoethvl)GSH
S_r2-(N7-auanvl)ethvl1GSH
Radioisotope
Diethylsulfate
Direct acting
N1"9''ethyladenine. N3»7-quanine. N3-cvtosine
Radioisotope
Dichlorvos
Direct acting
N7-methvlguanine and N9-fltethyladenine
Radioisotope
Oiethylnitrosamine (DEN)
Direct acting
N7. N3-ethy1guanine and N3-ethyladenine
Radioisotope
Direct acting
04-ethylthymi d i ne
Radioisotope
1.2-Dimethvlhvdrazine (SDMO
Methyldiazonium ion
N7-methylguanine and 06-*nethylquanine
Absorbance
1.2-0imethylhvdrazine (SDMH)
Methyldiazonium ion
Ull
lit*
84
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
— In vitro N/A 38.80
In vitro
N/A
38.80
In vitro
N/A
38.80
N/A
In vitro
N/A
38.80
In vitro
N/A
38.80
2 nM: 618 pmol/mq DNA
In vitro
N/A
160
In vitro
N/A
145. 196
Rat
Combined orqans
236
50 im/kq: 31. 3. 4 unol/mol DMA
T 1/2 ~ 50
Rat
Liver
57
40 dud DEN in H70 (80) 5.0 cmol/umol dT
T 1/2 ~ 11D
Rat
Liver
184
50 Don SDHH in HT0 (280) 700. 15 pnol/mq ONA
Rat
Non parenchymal cells 27
50 Dim SOMH in H,0 (28D) 800. 1 Dfnol/mq ONA
Rat
Heoatocvtes
27
85
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
MEASUREMENT TECH
1.2-Oimethvlhvdrazine (SOW) Methyldiazonium ion O^-methylthvmidine. Q6-methvlquanine Radioisotope
1.2-Oimethvlhydrazine (SOfffl)
Methyldiazonium ion
5 adducts
*2P dostlabelinq
1.2-Oimethvlhvdrazine (SOM)
Methyldiazoniun ion
N 7-.0*-methylquani ne
Radioisotope
1.2-Oimethvlhydrazine (SDMI)
Methyldiazonium ion
MU
Radioisotope
1.2-Oimethvlhvdrazine (SDfW)
Methyldiazonium ion
06-methylquanine
Fluorescence
1.2-Dimethvlhydrazine (SOW)
Methyldiazonium ion
N7-methvlauanine
Fluorescence
1.2-Oimethvlhvdrazine (SDMH)
Methyldiazonium ion
N7 -methylquanine
Fluorescence
1.2-Oimethvlhydrazine (SDMH)
Methyldiazonium ion
7-Methylquanine. 0s-methylguanine
Absorbance
¦ ¦
MM
KM
nn
Dimethylnitrosamine
Direct actina
N7-,06-methylquanine
saP postlabelinq
Dimethylnitrosamine
Direct actinq
N7. N9. 06-methyquanine. Na. N'-methyladenine
Radioisotope
Dimethylsulfate
Direct actinq
NjOlmethvlquanine. Ns-methyladenine
Radioisotope
Dimethylsulfate
Direct actino
N7.N3.06-methylguanine. N7.Nx.N3-methvladeine
Radioisotope
Dimethylsulfate
Oirect actina
N 7.06-methv1guan i ne. N 3-methvladen i ne
Radioisotope
Dimethylsulfate
Direct actina
Ns.N7,06-methylquanine.N1.N3.N7-methvladenine
Radioisotope
Dinitrosopiperazine Direct acting Yes Radioisotope
86
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF
20 nw/kq; 3.54. 494 pmol/mq DMA T 1/2 ~ 30h. ~ 40 hr Rat Liver 184
?: one adduct/<104 nucleotides = House Liver 182
20 mo/kg: 9.1-34.8 unol/mol guanine (0*H Gua) — House (ICR/Ha) Intestine 99
20 nw/kq: 8.2-23.0 unol/mol guanine (0*H Gua) = House (C57BL/Ha) Intestine 99
21 mq/kg: weekly for 14 weeks. 5-40 umol/mol Gua House Kidney 91
21 mq/ko: weekly for 14 weeks. 200-1100 mnol Gua — House Liver 91
21 mq/kq; weekly for 14 weeks. *- 30-70 umol/mol Gua — House Kidney 92
30 ppm. in HjO: 1000. SO pmol/mq QUA T 1/2 > 30 D ~ 1SD Rat Nonparenchvmal cells 28
^ ; 1200. 30 pmol/nq DMA T 1/2 > 30 D ~ 20 Rat Hepatocytes 28
150 ma/kq: one adduct/<104 nucleotides — House Liver 182
10 mq/kq: 379. 2. 37. 9. ~1 umol/mol DHA ID < T 1/2 < 6D Rat Liver 57
O.OflnW: 86. 10 umol/mol DMA T 1/2 -18. 3h V79 cell N/A 46
?: 3. 79. 1. 0.4. 0.4. 0.6. 16. umol/mol DMA — Balb C mouse Spleen cells 7?
15 uo/ml 92. 0.5. 12. umol/mol DMA V79 cell N/A 156
—- In vitro M/A 156
Human Bronchus, colon cells TJ_
87
-------
COMPOUND
ACTIVE METABOLITES
AODUCT STRUCTURE
MEASUREMENT TECH
Eoichlorohvdrin
Direct actinq
N7.06-alkylquanine Radioisotope
Ethionine
•?
N7-ethv1 quanine Radioisotope
Ethionine
?
MM UU
Ethionine
?
MM nu
Ethvlmethanesulfonate
Direct actinq
N7-06-methvlquanine —
Ethylmethanesulfonate
Direct actinq
N7.N3-methvlquanine.N7.N3-ethvladenine Radioisotope
Ethvlmethanesulfonate
Direct actinq
Ethvlated DNA
Ethvlmethanesulfonate
Direct actinq
um aw
Ethvlnitrosoquanidine
Direct actinq
M1 .M3 ,N7-ethvladenine.N3 ,N7-ethvlquanine. —
N3-ethvl-cvtosine. and Na-ethvl(uridine or thymine)
Ethvlnitrosourea
Direct actinq
M1.M3.M7-ethvladenine.H3.N7-ethvlquanine. —
N3-ethvl-cvtosine. and Nsethvl(uridine or thvamine)
Ethvlnitrosourea
Direct actinq
N7.0*-ethylquanine,02-cytosine.02.04 thymine Radioisotope
Ethvlnitrosourea
Direct actinq
M7-ethvlquanine.02-ethylthymine. and Oa-ethvlcvtosine ""
Ethvlnitrosourea
Direct actinq
O'-ethvlauanine Immunoassay
Ethvlnitrosourea
Direct actinq
"" Immunoassay
-------
DOSE/RESPONSE PERSISTENCE SPECIES TISSUES REF_
— — Rodents — 26
Nonlinear 31-500 mo/kg: 120-3800 pmol/mq DMA — Rat Liver 130
Nonlinear 31-500 mq/ko: 40-050 pmol/mq RNA = Rat Liver 130
Nonlinear 31-500 mq/kq: 1100-10000 pmol/mol protein — Rat Liver 130
= -- Rat — 26
200 mq/ko: 48. 1. ~ 1. 2. umol/mol QUA — Rat Liver 57
Nonlinear 2.5-50n#1 0.8-8.6 ethvlations/104 nucleotides — Yeast (neurospora crassa) N/A 221
Nonlinear 2.5-50n#1 0.6-6.7 ethvlations/104 nucleotides — Yeast (saccharomvces cerevisiae) N/A 222
— — In vitro N/A 196
In vitro
N/A
196
Mantnalian cells
N/A
196
— T 1/2 30-55 hr
Rat
Liver
196
— T 1/2 40. 50. 60 hrs
In vitro
N/A
153
Linear 0.1-200 uq/ml adduct 3x107 dGuo to 1 adduct/7.9x10s dCuo —
Rat
Linear 2.5-100 uq/q 1 adduct/2xlOs dGuo to 1 adduct/4xl04 dGuo —
Liver, brain
153
89
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
MEASUREMENT TECH
Ethyl nitrosourea Direct acting Na-ethyladenine. N7ethylquanine.Q6-ethylguanine Radioisotope
Ethylnitrosourea
Direct acting
06-ethylguanine
Radioisotope
UN
MM
N7-ethylguanine
M M
l-(2-Fluoroethyl)-3-
Direct acting
7-hydroxv ethylguanine and 7-chloroethylguanine
Radioisotope
cvdohexv1-1-nitrosourea
Glvcidaldehvde
Direct acting
N7.0*-methvlguanine
Gvrcmitrin
Decomposition products
N7-methylquanine
Radioisotope
Hydrazine
Tetraformvltrisazine
0*M7-methylquanine
Hethylmethane sulfonate
Direct acting
N7-methylguanine
MS
Methylmethane sulfonate
Direct acting
hh
MM
Hethvlmethane sulfonate
Direct acting
N7.06-methylquanine
Hethylmethane sulfonate
Direct acting
Na-methyladenine and N7-methylguanine
4-(methylnitrosamino)-l-
4-(methylnitrosamino)-l-
06-methylquanine
Immunoassay
(3-pvridyl)-l-butanone
(3-ovridvl)-l-butan-l-ol
MM
UN
MM
MM
MM
M M
MM
M M
90
-------
DOSE/RESPONSE
PERSISTENCE
SPECIES
TISSUES REF
78X0. 34X0. 5XH0-H22
Chinese hamster ovary cells
71
10 ira/ka: 1.31 adducts/10* quanine residues
ID < T 1/2 < 3D
House
Testis 190
"" : 1.79 adducts/106 quanine residues
1 D < T 1/2 < 3D
WW
190
In vitro
N/A 80
Rat
— 26
Rat
Liver, lunq 86
Rat
Liver 128
50 ira/kq: 12 uq/24 hr
N/A
Rat
Urine 65
SO mq/kg: 22 nq/10 mq qlobin
Rat
Globin 65
Rat
— 26
T 1/2 2.5. 11 H
Chlamydomonas
N/A 207
482 ranol/ml (1.3.24 hr culture time): 39. 65.
Cultured rat nasal mucosa
42
and 138 unol/mole quanine
87 im/kq: 219.13.34 unol/mole quanine
Rat
Nasal mucosa, lunq liver 42
40 ira/kq: daily for 140: 8.11 umol/mole quanine
Rat
Nasal mucosa, lunq 42
91
-------
COMPOUND
ACTIVE HETABOL1TES ADDUCT STRUCTURE
MEASUREMENT TECH
N-methvl-M-n i t rosourea
Direct actinq
N7,0A-methy1quanine.3-
-------
DOSE/RESPONSE
PERSISTENCE
SPECIES
TISSUES
REF
8.7 uQ/ml:?
55X0. 2X1. 84X0
Chinese hamster ovarv cells
71
0.5 mq/rat: 252. 2. 29. 1. 13. 6.5. 17 adducts
H0-H20
Rat
Urothelium
107
10® nucleotides
T 1/2 ~ 2.1.6.3.1.1.>210
50 mq/ko: 22 uo/24 hr
Rat
Urine
65
50 mq/kq: 2 no/10 mq qlobin
Rat
Globin
65
?: 1 adduct/< 104 nucleotides
House
Liver
182
Linear 0.08-0.6mH: 15-120 umol N7-methylquanine/mol DMA
House
Lymphoid cells
79
Linear 0.08-lirM: 15-120 umol N'-4nethvlQuanine/mol DNA
Human
Cells
60 uq/ml 3 hr: 102, 14. 6.2 umol/mol DNA
V79 cell culture
156
16 and 100 mq/kq; 0.8-7.0 umol/mole quanine
T 1/2 = 5.5H
House
Liver
53
16 and 100 mq/kq; 1.05-66 umol/mole quanine
T 1/2 = 5.2H
House
Kidney
53
0.B7 rnnol/kq; 242. 24 umol/mol ONA
Rat
Kidnev
53
In vitro
N/A
80
In vitro
N/A
131
Bindinq to RNA. DMA. soluble protein
146
93
-------
COMPOUND
ACTIVE METABOLITES
ADDUCT STRUCTURE
MEASUREMENT TECH
Nitroso-2.6-dimethyl Direct acting -- Radioisotope
rv—-"J ¦ . .... ..
Murpholine
H U
N-Nitrosodi-n-propyl amine
Direct actinq
7-n-propyl quanine
Nitroso(2-hydroxvpropyl)(2-
Direct actinq
•I M
oxooroDvl)-ami ne
Direct actinq
u n
Nitrosomethylethylamine
Direct actinq
M H
Nitrosobis(2-oxyopropyl) amine
Direct actinq
U M
N-fli troso-bi s-C2-oxopropy1) ami ne
Direct actinq
N7-methylquanine
Z-ethyl-ONN-azoxvmethane
Direct actinq
tl U
Z-methyl -ONN-azoxvmethane
Direct actinq
u u
N i trosofo is(2-hydroxypropy1)ami ne
Direct actinq
WW
N-n i trosanethyl benzyl ami ne
Direct actinq
N7.06-methylquanine
U H
N-ni trosanethylbenzylami ne
Direct actinq
nil
MM
N-nitrosomethylbenzylamine
Direct actinq
na
MM
N-Nitrosomethylbenzvl amine
Direct actinq
N7-methylquanine
Radioisotope
N-Ni trosanethyl(4-methylbenzyl)amine
Direct actinq
nu
u u
94
-------
OOSE/RESPOMSE PERSISTENCE SPECIES TISSUES REF.
" = Rat, hamster Liver 134
" — Rat, hamster Liver 134
" Rat Liver 9
22 Rat, hamster Liver 134
^ 22 Rat, hamster Liver 134
^ -- Rat, hamster Liver 134
^ — Rat, hamster Liver 134
__ Hamster Liver, lung, pancreas 129
^ — Rat, hamster Liver 134
¦¦ — Rat, hamster Liver 134
» -- Rat, hamster Liver 134
0.017nmol/fcq; 344.46 umol/mol guanine 2Z Bit Esophagus 94
0.017mno1/kq; 120.4.9 umol/mol guanine 2= Liver 24
0.017mno1/ltq; 65.7.7 umol/mpl guanine 2= lung 94
0.017 tnmol/kq; 10.3 umol/mol ONA — Rat Forestomach 94
0.017 mnol/kq: 22.4 umol/mol QUA 2= Eat Esophagus 94
95
-------
COMPOUND
ACTIVE METABOLITES AOOUCT STRUCTURE
MEASUREMENT TECH
MM
Direct actinq
MM
MM
Direct actinq
Mil
N U
N-Nitroso-N-methyl-W'-nitroquanidine
Direct actinq
0®.N7-methylquanine
N-Nitroso-N-methylurethane
Direct actinq
u u
Nitrosopioeridine
Direct actinq
Yes
NitrosoDvrroline
Direct actinq
Yes
N-(2-oxopropy1)-N-n i trosourea
Direct actinq
0<,N7-methylquanine.N3-fliethy1adenine
MS
N-Nitroso 2-oxopropylamine
Direct actinq
06.N7-methylquanine
UV absorbance
1-n-oroDvl-l-nitrosourea
Direct actinq
06.N7-n-propvlquanine.O#.N7-iso-propylquanine
Radioisotope
B-oroprolactone
Direct actinq
0®. N 7 -al k vl quan i ne
1.3-oropane sultone
Direct actinq
N7 .N1.0®-alkylquanine
UV absorbance
Propylene oxide
Direct actinq
Ns-(2'-Hydroxypropvl)histidine
GC-MS
Streptozotocin
Direct actinq
N7.06-methylquanine. N7.Ns-methyladenine
Radioisotope
MM
Direct actinq
UU
UU
MM
Direct actinq
MM
MU
MM
Direct actinq
MM
MU
96
-------
DOSE/RESPONSE
PERSISTENCE
SPECIES
TISSUES
REF
0.017 mnol/kq: 30.8 u>nol/mol DMA
Rat
Liver
94
0.017 mnol/kq: 13.6 umol/mol DNA
Rat
Lunq
94
Rodents
26
Rodents
26
Cultured, human cells
77
Cultured, human cells
77
Nonlinear. 10-40nfl: ~25-60nmol/N7-JteGua/mq DNA
In vitro
N/A
131
2.0nmo1/kq: 55. 760 unol/mol quanine
Rat
Liver
132
InM. 68 pmol alkylation/mq DNA
In vitro
N/A
151
Rodents
26
In vitro
N/A
88
"Low" to 10 Dom: 0.9-13 ntnol/q Hb
Human
Hemoqlobin
21 mq/kq: 486.21.16.35 omol/umol quanine
Rat
Liver
34
21 mq/kq: 289.11.13.17 pmol/umol quanine
Rat
Kidney
34
21 mq/kq; 96.9.6.6 umol/umol quanine
Rat
Intestine
34
21 mq/kq: 5.<1.<1.<1 Dmol/unol quanine
_
Rat
Brain
34
97
-------
COMPOUND ACTIVE METABOLITES ADPUCT STRUCTURE MEASUREMENT TECH
" Direct acting ™^
" Direct acting 5 adducts present 32P-postlabeling
Vinyl chloride Chlorethvlene oxide and 3.N4-ethenocvtidine l.N6-ethenoadenosine. and —
chl oroaceta 1 dehyde l.M2-ethenoquanosine
S-viny 1 homosvsteine Same as ethionine
98
-------
OOSE/RESPOMSE PERSISTENCE SPECIES TISSUES HEP
21 mq/kq; 43.2.3.2 pmol/umol guanine — Rat Pancreas 34
?; 1 adduct/<104 nucleotides — House Liver 182
— In vitro M/A 192.196
99
-------
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107
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69
70
71
72
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74
75
76,
77
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REFERENCES (Continued)
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AU Harris, C.C.
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108
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