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Some Current Perspectives on Chemical Carcinogenesis
Experimental Animals: Presidential Address1
Elizabeth
McArdle
in Humans and
C. Miller
Laboratory
lor Cancer Research,
University
of Wisconsin
Medical Center, Madison,
Wisconsin
53706
Knowledge in chemical carcinogenesis now spans many
disciplines and is so large a subject that many areas can be
considered here only briefly or not at all. Similarly, in many
cases references to reviews have been substituted for
references to the original literature in order to keep the
bibliography within manageable limits.
Environmental
I am very honored to have the opportunity to discuss
some aspects of chemical carcinogenesis with you tonight.
Those of you who know my husband, James A. Miller, and
me could not have been surprised at my choice of this
subject, for we have spent an exciting 35 years together in
trying to ferret out some of the properties of chemical
carcinogens and the changes that they cause in their target
tissues. When we started our work together in 1942, chem
ical carcinogenesis was a rather limited field that had
attracted only a small number of investigators. During the
intervening years interest in chemical carcinogenesis has
grown markedly and, like the scientific disciplines of or
ganic chemistry, biochemistry, and molecular biology, on
which it depends, has grown greatly in its sophistication.
1 Presented on May 18, 1977. at the 68th Annual Meeting of the American
Association for Cancer Research, Denver, Colo.
Chemical Carcinogens
for the Human
The great interest in chemical carcinogenesis among
both scientific and lay persons is based in part on the
conclusion of epidemiologists, starting with Higginson (78)
in 1969, that 60 to 90% of human cancers have important
environmental factors in their etiologies. This deduction is
based primarily on the large differences in incidences of
specific cancers, usually measured by mortality figures,
from country to country and even within countries (see also
Refs. 41 and 42). As shown by Haenszel and his collabora
tors (65, 66) and others, these differences in geographic
incidences are not primarily genetically determined. Thus,
the cancer patterns for migrants from one country to
another, and especially those of their children, generally
change from those characteristic for inhabitants of the
mother country toward those characteristic of the inhabit
ants of their new country.
Other than skin cancer, for which solar UV is an important
causative factor (44, 212), emphasis has been placed on
environmental chemicals as major factors in the causation
of human cancer. This emphasis has resulted from the lack
of definitive data on the roles of infectious viruses in the
causation of human cancers (70),2 the indication that ioniz
ing radiations play only a relatively minor role in the overall
cancer incidences (88), the fact that over a dozen specific
chemicals have been identified as causes of some human
cancers (Table 1), and the conclusion that a high proportion
of all human lung cancers is associated with cigarette
smoking (191). In addition to the chemicals generally rec
ognized as carcinogens in humans as a result of industrial,
medical, and societal exposures, a number of other chemi
cals in the environment, such as aflatoxin B, and certain Nnitrosamines and N-nitrosamides, are strongly suspected
of causing cancers in humans (13, 84, 119, 216, 231). It
appears very likely that additional chemical carcinogens of
both natural and synthetic origin will be identified as causes
of human cancer.
2 There is no unanimity of opinion on the possible roles of viruses in the
causation of human cancer. While most investigators today appear to find
the available data unconvincing as evidence for an important role of
infectious viruses, Gross (60) believes that viruses may be key factors in the
etiology of human cancer.
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E. C. Miller
Table 1
Chemicals generally recognized as carcinogens in the human
ChemicalIndustrial
formationUrinary
of tumor
exposures2
/3)-NaphthylamineBenzidine
(or
(4,4'-diaminobiphenyl)4-Aminobiphenyl
4-nitrobiphenylBis(chloromethyl)
and
etherBis(2-chloroethyl)
sulfideVinyl
chlorideCertain
oilsChromium
soots, tars,
compoundsNickel
compoundsAsbestosAsbestos
bladderUrinary
bladderUrinary
bladderLungsRespiratory
tractLiver
mesenchymeSkin,
lungsLungsLungs,
484, Vol.
184,Vol.
and84,
Vols. 1
484, Vol.
984, Vol.
784, Vol.
10384,
Vol. 3;
Vol.284,
and84,
Vols. 2
and84,
Vols. 2
and84,
Vols. 2
sinusesPleura,
nasal
peritoneumLungs,
smokingMedical
plus cigarette
pleura,
perito
neumUrinary
exposuresA/,/V-Bis(2-chloroethyl)-2-naphthylamine(Chlornapthazine)DiethylstilbestrolSocietalCigarette
Vol.484,
bladderVaginaLungs,
61911514111414
Vol.
smokeBetel
nut and tobacco
quidsSites
urinary tract, pan
creasBuccal
mucosaRef.84,
Early Studies
The beginnings of our knowledge on chemical carcinogenesis can be traced to two observations in London,
England. The first was that of the astute physician John Hill
(172) in 1761 on the development of nasal cancer as a
consequence of excessive use of tobacco snuff, and the
second was that of the perceptive surgeon Percival Pott
(164) on the unusually high incidence of cancer of the skin
of the scrotum among young men who were chimney
sweeps in their childhood. Pott's observation was appar
ently the basis of the first preventive measures against
chemically induced cancers in humans, since, according to
Clemmesen (33), 3 years later the Danish chimney swee
per's guild urged its members to take daily baths. About
100 years later Butlin (27), another English physician, con
cluded that the low incidence of scrotal cancer among the
chimney sweeps in northern Europe as compared to the
high incidence among English chimney sweeps was related
to the better personal hygiene and protective clothing of
the former group.
The development of skin cancer in certain workers was
shown by von Volkmann in Germany in 1875 and by Bell in
Scotland in 1876 to be associated with contact of the skin
with tar and paraffin oils that, as we now know, contained
polycyclic aromatic hydrocarbons (reviewed in Ref. 64).
The latter observations led in 1907 to the inclusion of such
skin cancers in the third schedule of the British Workmen's
Compensation Act (75). These observations were followed
in the latter part of the 19th century by Rehn's acute
observations (173) on the development of cancer of the
urinary bladder in three workers in a so-called "aniline" dye
factory in Germany and by the subsequent observations in
many countries on the association between human bladder
cancer and occupations that resulted in relatively gross
exposures to 2 (or /3)-naphthylamine, benzidine (4,4'-diaminobiphenyl), or 4-aminobiphenyl (Ref. 29, pp. 40-45).
These observations on higher incidences of specific can
cers in individuals with particular chemical exposures
1480
stimulated attempts to induce tumors in experimental ani
mals by application of the implicated chemicals and related
substances. Fischer (52) met with some success in 1906,
when he found that the application of the azo dye scarlet
red (1-[4-(o-tolylazo)-o-tolylazo]-2-naphthol)
induced a proliferative lesion of the skin in rabbits. However, these
lesions did not progress to frank neoplasia and regressed
after the applications of scarlet red were stopped. A number
of investigators sought to demonstrate the carcinogenic
activity of soots and tars in experimental animals. Success
was achieved in 1915 by Yamagiwa and Ichikawa in Japan
who induced carcinomas on the ears of rabbits by repeated
topical applications of coal tar for long periods (see Ref.
64). Tsutsui in 1918 then showed that tars are also carcino
genic for mouse skin, and in 1922 Passey induced cancer in
mouse skin by application of ether extracts of tars (64).
The induction of skin cancer with tars and extracts
thereof led to searches for the active agents. Chemical
studies by Bloch and Dreifuss and their collaborators in
Germany suggested that polycyclic aromatic hydrocarbons
were the active materials (64). More conclusive evidence
was available when Kennaway (96) in 1925 produced carci
nogenic tars by pyrolysis in a hydrogen atmosphere of
several organic materials, including acetylene. Hieger's
work (77) revealed that the fluorescence spectra of products
from the tars and of synthetic benz(a)anthracene derivatives
were similar. This observation led to the demonstration by
Kennaway and Hieger (97) in 1930 of 1,2,5,6-dibenzanthracene3 or, as it is now known, dibenz(a,h)anthracene, as the
first synthetic carcinogen (Chart 1). Soon thereafter, a
carcinogenic hydrocarbon isolated from coal tar was iden
tified by Cook, Hewett, and Hieger (35) as 3,4-benzpyrene,
now called benzo(a)pyrene. Extensive studies, especially by
Kennaway and his associates in England and by Shear,
Fieser, and their associates in this country, soon led to a
3The hydrocarbon was designated as 1,2,7,8-dibenzanthracene in the
original paper (97). According to Hartwell (Ref. 69, p. 238) it should have
been called 1,2,5,6-dibenzanthracene.
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rororc? «W
DIBENZIo.hlANTHRACENE
CARBON
TETRACHLORlOe
2-ACETYLAMINOFLUORENE
(N-2-FLUORENYLACETAMIDE)
BENZOfolPYRENE
B>O
BERYLLIUM
ETHYL
3-METHYLCHOLANTHRENE
7,12-DIMETHYLBENZIolANTHRACENE
,CHj
2',3-OIMETHYL-4-AMINOAZOBENZENE
OXIDE
CARBAMATE
N.N-DIMETHYL-4-AMINOAZOBENZENE
CH3-N
CHo-CHo-CI
'
1CH2-CH2-O
N-METHYL-BIS(ß-CHLOROETHYD-
CHj
CHj'
N-NO
DIMETHYLNITROSAMINE
AMINE
CHj-Q^-S-CHj-CHj-
CH- COOH
NH2
Chart 2. The structures of some chemical carcinogens identified between
1940 and 1960.
Chart 1. The structures of the principal chemical carcinogens discovered
prior to 1940.
large literature on the chemical features required for the
carcinogenicity of the polycyclic aromatic hydrocarbons
(reviewed in Ref. 29, pp. 137-164; Ref. 40). Of the carcino
genic polycyclic aromatic hydrocarbons studied during this
early period, benzo(a)pyrene, 3-methylcholanthrene, dibenz(a,tì)anthracene, and 7,12-dimethylbenz(a)anthracene
have been most widely used in subsequent experimental
studies.
Following the early work of Fischer, Yoshida (235)
showed in 1933 that p.o. administration of a derivative of
scarlet red, o-aminoazotoluene or 2',3-dimethyl-4-aminoazobenzene, induced liver tumors in rats and mice. Three
years later Kinosita (102) demonstrated the strong carcino
genicity of an isomer, /N/,/V-dimethyl-4-aminoazobenzene. In
1938 Hueper, Wiley, and Wolfe (82) succeeded in the
induction of cancer of the urinary bladder in dogs fed 2naphthylamine. Thus, by 1940 the epidemiological data on
the carcinogenicity of an aromatic amine and of coal tars
and soots for man had been complemented by definitive
data on the carcinogenicity of pure chemicals contained in
these mixtures for laboratory animals. Furthermore, in 1932
Lacassagne (109) made the first observations on the devel
opment of mammary cancer in male mice treated with
estrone and thus opened the large field of hormone-in
duced tumors for experimental study.
This limited list of known carcinogenic chemicals ex
panded markedly in the 1940's (Chart 2). The carcinogenic
ity of 2-acetylaminofluorene was first observed by Wilson,
DeEds, and Cox (228) in 1941. Subsequent studies showed
the versatility of its carcinogenic activity for various tissues
and species (223), and a number of related amides were
found to have similar activity (138, 139, 143). Also in 1941
Edwards (43) reported the induction of hepatomas in mice
by carbon tetrachloride; a number of halogenated hydro
carbons have since shown similar activity. Urethan (ethyl
carbamate) was found by Nettleship and Henshaw (153) to
induce adenomas of the lung in mice, and subsequent
studies demonstrated the versatility of this carcinogen
(146). The induction in 1946 of osteosarcoma in rabbits by
zinc beryllium silicate and beryllium oxide by Gardner and
Heslington (57) was the first experimental demonstration of
the carcinogenicity of certain inorganic chemicals (Ref. 29,
pp. 113-134). Similarly, the carcinogenicities of thiourea,
thioacetamide, and the nitrogen mustards were first ob
served in this decade (20, 53, 168).
Data reported in the 1950's revealed the carcinogenic
activities of new classes of chemicals: the wide range of
alkylating agents (112); the dialkylnitrosamines (119); ethionine (48); and the pyrrolizidine alkaloids (34, 183). The
carcinogenicity of the pyrrolizidine alkaloids, like that of
estrone, made it evident that chemical carcinogens are not
solely the products of chemists or of high-temperature
combustions. A number of metabolites of plants and micro
organisms are now known to be carcinogenic for experi
mental animals (Chart 3) (142), and it is probable that many
more naturally occurring carcinogens exist among the vast
number of uncharacterized nonnutrient metabolites of liv
ing cells. Some of these can be expected to contact human
tissues, usually in low doses, through food or as products
of the intestinal flora.
The large number and great variety of organic chemical
carcinogens now known belong to over a dozen different
classes. A much smaller number of inorganic chemical
carcinogens have been identified, and many classes of
inorganic chemicals remain to be tested for their carcino
genic potentials (69, 84, 91, 185, 208).
An achievement of great importance was the develop
ment of techniques for the malignant transformation of
cells in culture by chemicals. The first reproducible system
for malignant transformation was reported in 1963 by Berwald and Sachs (12), who used hamster embryo cells. The
maintenance and transformation of rodent fibroblast cul
tures have since been examined in considerable detail.
Systems for the transformation of epithelial cells have been
more difficult to develop. This important area of research,
which has provided valuable techniques for elucidation of
the molecular events that occur during chemical carcinogenesis, has been critically reviewed by Heidelberger (73).
Tumor Induction as a Multistage
Phenomenon
Skin. The classical methods for the induction of tumors
by chemicals have usually involved the administration of a
single agent, most often in a repetitive manner, with the
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E. C. Miller
HOHjC
OH
CYCASIN
(CYCAD TREE FERNS)
R'-O
CH0-0-C-R
b'
0-CH3
AFLATOXIN
(ASPERGILLUS
B,
PYRROLIZIDINE ALKALOIDS
(SENECIO,CROTOLARIA AND
HELIOTROPIUM GENERA)
FLAVUS)
OCHj
CI
MITOMYCIN
(STREPTOMYCES
C
CAESPITOSUS)
CH3
GRISEOFULVIN
(PENICILLIUM
GRISEOFULVUM)
Chart 3. The structures of some chemical carcinogens that are products
of plants and microorganisms.
end point being the development of benign or malignant
tumors or both. However, it has been evident for many
years that the induction of skin tumors in the mouse and
rabbit is a multistage process.
At first shown by Rous and his associates (55, 179),
Mottram (150), and Berenblum (9) and further developed by
Berenblum and Shubik, Boutwell, and Van Duuren and
their associates, the administration of a limited dose of a
chemical carcinogen to mouse or rabbit skin causes
changes in some cells that are imperceptible in the absence
of further treatment and do not by themselves result in tu
mors (reviewed in Refs. 16, 17, and 213). This initiation can
be effected by a single dose of the carcinogen, such as an
alkylating agent, polycyclic aromatic hydrocarbon, or ethyl
carbamate. Initiation is generally considered to be com
pleted rapidly and to be essentially irreversible. The sec
ond stage, promotion, occurs over a period of weeks and
months and is, at least in its early phases, largely reversible.
The classical promoting agent for mouse skin is croton oil
from the seeds of Croton tiglium; it was first used by
Berenblum as a cocarcinogen (9). Structural characteriza
tion of the active ingredients of the croton oil as 12,13diesters of the diterpene alcohol phorbol (Chart 4) was
accomplished through independent studies in several lab
oratories, especially those of Hecker and of Van Duuren
(see Refs. 71 and 213). Of these diesters tetradecanoylphorbol acetate is by far the most active; anthralin (1,8-dihydroxy-9-anthrone), which is much less active as a promoter
than the latter ester, is the most active of the nonphorbol
derivatives studied (213).
Chart 5 illustrates some essential features of the mouse
skin tumor initiation-promotion system. Administration of
only a single small dose of an initiator such as 7,12-dimethylbenz(a)anthracene or of only repetitive doses of the pro
moter tetradecanoylphorbol acetate does not lead to gross
tumors (16, 213). However, sequential administration of the
hydrocarbon and then repeated doses of the phorbol ester
give rise to a large number of papillomas within 3 to 4
months and carcinomas in about 1 year. Similar results are
obtained even if several months elapse between the appli
cation of the initiator and the first dose of promoter (11,
215). Important facts are that the sequence of application
1482
Chart 4. The structure of 12-O-tetradecanoylphorbol-13-acetate or phorbol-12-myristate-13-acetate, a very potent promoter for tumorigenesis in
mouse skin. This promoting agent is found in the seeds of Croton tiglium.
0
-TIME
I = INITIATOR
P-
.single dose)
PROMOTER i many doses)
Chart 5. Diagrammatic representation of the two-stage induction of tu
mors in the skin of the mouse. Typical initiators are the polycyclic aromatic
hydrocarbons, ethyl carbamate, and certain alkylating agents. The most
potent promoting agent is 12-O-tetradecanoylphorbol-13-acetate (Chart 4)
Papillomas develop within about 12 to 20 weeks, and carcinomas develop at
about 1 year.
of the initiator and promoter cannot be reversed and that
promotion requires repeated doses of the promoter.
Liver. About 20 years ago Weiler (219) observed islands
of morphologically normal hepatic cells in livers from rats
given N,W-dimethyl-4-aminoazobenzene
and then main
tained on a dye-free diet for relatively long periods. As
further elucidated by Hughes (83), these cells could be
distinguished from the majority of the hepatic cells by their
decreased ability to adsorb fluorescein-conjugated globu
lins, but they were often not recognized as different from
other liver cells on staining with hematoxylin and eosin.
The studies on such altered, possibly premalignant cells,
have been greatly extended by the groups working with
Farber, Becker, Friedrich-Freksa, Rabes, and Emmelot (see
Refs. 49, 50, 181, and 182). Farber, Becker, and their
colleagues have designed protocols for the development of
gross nodules of apparently nonneoplastic hepatic cells. In
rats subsequently maintained in the absence of the carcin
ogen, the cells of many of these hyperplastic nodules
ceased to proliferate and were reincorporated into more or
less normal hepatic structures (reviewed in Ref. 50).
A two-stage system of hepatic tumor formation was intro
duced in 1971 by Peraino and his associates (158, 159),
who used a limited period of administration of 2-acetylaminofluorene and subsequent long-term dosing with pheno-
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Current Perspectives on Chemical Carcinogenesis
barbital. The result of the combined treatment was a high
incidence of relatively highly differentiated hepatocellular
carcinomas, while the limited treatment with 2-acetylaminofluorene alone induced far fewer hepatic tumors. Longterm administration of phénobarbital to rats induced no
tumors (158, 159) or a low incidence after a long latent
period (178).
Modifications of these systems are now being examined
by many investigators. In preliminary studies Pitot (162)
obtained hepatocellular carcinomas 1 year after administra
tion of a single dose of diethylnitrosamine, 5 mg/kg, to
partially hepatectomized rats that were, after 2 months, fed
phénobarbital continuously in the diet. The livers of these
rats also contained large numbers of "foci" of hepatic
parenchymal cells that were distinguishable from normal
cells by histochemical, but not morphological, techniques
at the light microscope level. These groups of cells, which
contained as few as 500 cells, were recognized by their
deficiencies in histochemically detectable glucose-6-phosphatase or canalicular ATPase or by increases in y-glutamyltranspeptidase. The cells in these foci differed from
normal parenchymal cells in 1, 2, or all 3 of these enzymatic
activities (Chart 6), and the individuality of such cells will
probably become even more evident as further markers are
studied. Far fewer of these groups of altered cells were
observed in livers from rats given only the low dose of
diethylnitrosamine, and they were rarely seen in livers from
rats given only the phénobarbital. The broad diversity of
phenotype from the earliest recognizable altered cells to
the primary tumors and the elucidation of the precursorproduct relationships, if any, between these two classes of
cells will be exciting areas of research in the new few years.
Other Tissues. Although the models have been much
less well developed than for mouse skin tumors or rat liver
tumors, evidence has also been presented for multistage
induction of tumors of the mammary gland (4), thyroid (67),
lung (3), urinary bladder (25, 76), and certain other tissues.
Some of the problems in the interpretation of these data
have been discussed by Berenblum (10).
The two stages of initiation and promotion have also
been demonstrated in mouse fibroblast cultures. Thus, as
shown by Lasne and his associates (110) and by Mondai ef
al. (148), a single short treatment with less than 1 ^g of a
polycyclic hydrocarbon did not give rise to clones of trans
formed cells. However, the subsequent exposure of the
cells to 12-tetradecanoylphorbol-13-acetate
gave rise to
large numbers of clones of transformed cells. The number
of transformed cells was dependent both on the amount
and structure of the initiating agent and on the structure of
the promoting agent.
Electrophilicity
ogens
as a Common Property of Ultimate Carcin
Several observations early suggested that the metabolism
of chemical carcinogens might be a key factor in their
carcinogenic activities. Thus, as the number and variety of
chemical carcinogens increased, it became more and more
evident that these chemicals lacked a common structural
feature (Charts 1 to 3). Furthermore, some carcinogens,
especially the aromatic amine derivatives, produced tumors
at distant sites such as the liver and urinary bladder regard
less of the route of administration. An early clue to the
possible metabolic activation of a carcinogen was our
finding in 1947 of the covalent binding of a metabolite of
/V,/V-dimethyl-4-aminoazobenzene to the hepatic proteins
of rats fed this dye (131). Similar observations on the
formation in target tissues of protein-bound derivatives of
the polycyclic aromatic hydrocarbons and 2-acetylaminofluorene soon followed in our laboratory and those of
Heidelberger and the Weisburgers (reviewed in Refs. 72,
74, 132, 133, and 223).
Other studies, especially in the laboratories of Heidelber
ger (74, 206), Ketterer (98), and Sorof (192) and their
associates, showed marked specificities of certain proteins
of the target tissues for binding to these carcinogens. The
reasons for these specificities and the roles of these pro
tein-bound carcinogen derivatives in carcinogenesis have
not been elucidated, but Mainigi and Sorof (121) have
recently reported data consistent with their suggestion that
the principal hepatic azo protein is a vehicle for the intracellular transport of an ultimate carcinogenic derivative of
3'-methyl-/V,/\/-dimethyl-4-aminoazobenzene.
The recognition of the central roles of DNA's as store
houses of genetic information and of RNA's in the transla
tion of the genetic information for the synthesis of cellular
proteins provided new perspectives on the possible roles of
nucleic acid-carcinogen adducts in carcinogenesis and
provided an impetus to search for such derivatives. Wheeler
and Skipper (226) reported in 1957 that 14Cfrom [methyl14C]bis(2-chloroethyl)methylamine was incorporated into
the purine fractions from the RNA and DMA of certain
mouse tissues. Subsequent studies by Farber and Magee
and their colleagues (51, 118, 123), by Brookes and Lawley
(24), by Heidelberger (72), and by Stekol ef al. (195) soon
demonstrated the incorporation of 14C from 14C-labeled
ethionine, 2-acetylaminofluorene,
dimethylnitrosamine,
and polycyclic hydrocarbons into the DNA and RNA of the
target tissues. Since that time the administration of all
carcinogens that have been adequately studied has yielded
DNA-, RNA-, and protein-bound derivatives in the target
tissues (74, 133). Correlations between the levels of these
nucleic acid- and protein-bound derivatives and the likeli
hood of tumor development were obtained in many, but not
all, cases. Taken as a whole, the data indicated that macromolecular-bound forms of the carcinogens were a neces
sary, but not sufficient, correlate for the induction of tu
mors by chemical carcinogens.
Gradually, these and other studies led to the generaliza
tion that the great majority of chemical carcinogens were
active only after metabolism to ultimate carcinogens (;'.e.,
the derivatives that actually initiate the neoplastic event).
The known exceptions are the carcinogens that are alkylating or acylating agents per se. Further, the data then
available suggested to us that the ultimate forms of chemi
cal carcinogens might all be strong electrophilic reactants
(Chart 7) (141). This conclusion still appears to be valid,
although a few carcinogens, such as Adriamycin (122), may
be active through tight noncovalent binding rather than as
a result of covalent reaction with a macromolecule. Thus,
the known ultimate carcinogens contain relatively electrondeficient atoms that seek to react with nucleophilic sites,
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E. C. Miller
identification
of the specific role of each adduct in the
carcinogenic process.
Since carcinogenic
chemicals are promoters as well as
initiators,
it is possible that carcinogen-macromolecule
interactions are of fundamental importance in both phases
of the overall process. Furthermore, since there are initia
tors with little or no promoting activity (e.g., ethyl carbamate in mouse skin) (180, 213) and promoters with little or
no initiating activity (e.g., phorbol esters) (71,150, 213), the
ultimate initiating and ultimate promoting agents from a
given precarcinogen
may be either identical or different.
Furthermore, carcinogenesis
by a chemical that has both
initiating and promoting activities may require interaction
with more than one kind of macromolecule,
e.g., with both
DNA and specific proteins.
Examples of the Metabolic
Chemical Carcinogens
Activation
and Reactivity
of
Potential Donors of Simple Alkyl Groups. This group
includes especially the dialkylnitrosamines,
dialkylhydrazines, aryldialkyltriazenes,
and alkylnitrosamides.
The first
three classes of these versatile carcinogens are metabolically dealkylated by the mixed-function
oxidases in the
endoplasmic
reticulum,
and these monoalkyl derivatives
spontaneously
decompose to the corresponding
monoalkyldiazonium ions (112, 119) (Chart 9). The W-nitrosimides
and A/-nitrosamides
do not require enzymatic activation,
since their reaction with water and other cellular nucleo
philes results in the formation of the same alkylating interChart 6. Biochemical phenotypes of foci of altered parenchymal cells in a
section of liver from a rat given 1 p.o. dose (5 mg/kg) of diethylnitrosamine
24 hr after a partial hepatectomy and, starting 2 months later, 0.05%
phénobarbitalin the diet for 6 months. The enzyme activities were deter
mined on serial sections. O, glucose-6-phosphatase-deficient areas,
,
canalicular ATPase-deficient areas;
•y-glutamyltranspeptidase-positive areas. (This chart was kindly provided by Dr. H. C. Pitot of the McArdle
Laboratory.)
H
H
CELLULAR
la+---
NUCLEOPHILE
—ti
DISPLACED
ELECTROPHILE
^
ELECTROPHILIC REACTANT
(x+---y")
>bx
ELECTROPHILIC
ATOM
MET-SH
CYS*
LEAVING
NUCLEOPHILE
CI"2
PCû
1^N~,-0"
fromstrained
(HIS1N-I.N-3)/
GIN-3.N-7.N2)'^AtN-l
NONEORH*:S
N-7)jC(N-3)YH
N-3
ringsR-COj
TYRIC-3)
G
' (C-B)*C-OH
TYR6<°''l3=-OH
, HS04
DMA-C*>R-SCÕ.R-CO¿,HSO¿H
:NU =
9 _ , R-S
R-O-tf-0
O
R'
Chart 7. Examples of strong electrophihc reactants (positive ions or
uncharged molecules with electron-deficient atoms) and their reactions with
nucleophiles (:NU) through sharing of electron pairs of electron-rich atoms.
i.e., atoms that have easily shared electrons. These nucleo
philic sites are relatively abundant in DNA's, RNA's, and
proteins and include certain oxygen and nitrogen atoms in
the nucleic acids and nitrogen, sulfur, and oxygen atoms in
proteins (Chart 8). Because some, and probably many,
precarcinogens
are metabolized to more than one ultimate
carcinogen and because there are multiple nucleophilic
sites in each macromolecule,
multiple DNA-, RNA-, and
protein-bound
derivatives of each carcinogen are possible
and are frequently observed. Accordingly,
basic problems
of great importance
today are the elucidation
for each
carcinogen of those informational
macromolecule-bound
products that are important
in carcinogenesis
and the
1484
Chart 8. In vivo macromolecular nucleophilic targets of chemical carcin
ogens that have been identified up to the present.
DIMETHYLNITROSAMINE
N-METHYL-N-NITROSOUREA
N-NO
,c0
H2N
itHop,
non-enzymatic
rCH3|N=NfOH
*;
-i
J, DNA, RNA, PROTEIN
CHj-ONA, CHj-RNA WITH cf-CHj-G,
7-CH3-G,
3-CHj-A,
ETC.
CH3-PROTEIN WITH I- and î-CHj-HISTIDINE.S-CHj-CYSTEINE,
ETC.
Chart 9. The in vivo conversion of dimethylnitrosamine and of N-methylN-nitrosourea to a reactive electrophile and its reaction with cellular macromolecules.
CANCER
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VOL. 38
Current Perspectives on Chemical Carcinogenesis
mediates. Administration of these carcinogens results in
the alkylation, to various degrees, of a wide variety of
nucleophilic sites in the target cells.
2-Acetylaminofluorene. 2-Acetylaminofluorene is a more
complex carcinogen for which the metabolic activation has
been worked out in some detail for one target tissue (the
liver) (Chart 10). Studies with Cramer in our laboratory in
1960 showed that rats fed 2-acetylaminofluorene converted
it to a new metabolite, A/-hydroxy-2-acetylaminofluorene,
which is a stronger carcinogen than is the parent com
pound and which is also active in a wider range of tissues
and species (36, 136, 137, 140). Although administration of
both 2-acetylaminofluorene and its /V-hydroxy derivative
yielded nucleic acid- and protein-bound derivatives, espe
cially in the rat liver (107, 133, 223), these carcinogens are
not reactive in vitro and further metabolism seemed neces
sary. Studies by King and Phillips (101) and by DeBaun in
our laboratory (37) showed the presence of soluble sulfotransferase activity for N-hydroxy-2-acetylaminofluorene in
rat liver; the product of this reaction, /V-sulfonoxy-2-acetylaminofluorene, appears to be a major ultimate carcinogenic
metabolite in rat liver. Thus, hepatic sulfotransferase activ
ity under various conditions correlates with susceptibility to
hepatic tumor formation, the sulfuric acid ester is a very
strong electrophile, and the hepatotoxicity and hepatocarcinogenicity of W-hydroxy-2-acetylaminofluorene were de
creased on reduction of the amount of available active
sulfate or 3'-phosphoadenosine 5'-phosphosulfate in vivo
(37, 38, 224). The biological activity of the sulfuric acid
ester was also evident from its high mutagenic activity in a
DMA-transforming system (120).
In spite of the apparent major importance of the sulfuric
acid ester for liver tumor formation in the rat, it should be
noted that three other enzymatic pathways for conversion
of /V-hydroxy-2-acetylaminofluorene to electrophilic reactants have also been observed in rat liver (Chart 11). As
shown by Bartsch and Hecker (7), W-hydroxy-2-acetylaminofluorene undergoes a peroxidase-catalyzed one-electron
oxidation to yield a free nitroxide radical, and two of these
radicals can dismutate to yield the electrophiles /V-acetoxy2-acetylaminofluorene and 2-nitrosofluorene. An electron
spin resonance signal that is consistent with the formation
of this free radical has been observed by Stier ef a/. (196) on
incubation of 2-aminofluorene with rabbit liver microsomes.
Further, Bartsch in our laboratory (6) showed that rat liver
COCH,
rot liver
/COCHj
|XOH
E.R..,
+ NADPH+ OZ
2-ACETYLAMINOFLUORENE
(AAF)
N-HYDROXY-AAF
rot liver
cy totol
+ PAPS
AAF- RESIDUES COVALENTLY
BOUND TO HEPATIC
INFORMATIONAL MACROMOLECULES
PAPS •3-photphoadtnotlne
,COCH3
rot liver DNA,
RNA, protein
So-so,
AAF-N-SULFATE
-5-
photphoiulfati
("active «ulfot«")
Chart 10. The major pathway for the metabolic activation of 2-acetylami
nofluorene for carcinogenesis in rat liver. E.R., endoplasmic reticulum.
-C-CH
DISMUTATION*
N-ACETOXY-AAF
PEROXIDASE
2-NITROSOFLUORENE
H2
N-ACETOXY-AF
GLUCURONYL
TRANSFERASE
+ UDPGA
0-GLUCURONIDE
Chart 11. Pathways, in addition to the formation of the sulfuric acid ester,
for the metabolism of/V-hydroxy-2-acetylaminofluorene to electrophilic reactants in rat liver. N-ACETOXY-AAF, N-acetoxy-2-acetylaminofluorene; NACETOXY-AF, N-acetoxy-2-aminofluorene;
UDPGA, uridine diphosphoglucuronic acid.
cytosol forms the very potent electrophile /V-acetoxy-2-aminofluorene by enzymatic transfer of the acetyl group from
the nitrogen atom of /V-hydroxy-2-acetylaminofluorene to
the oxygen atom of the hydroxylamine. Finally, N-hydroxy2-acetylaminofluorene is converted by hepatic microsomes
to the weakly electrophilic O-glucuronide (86, 130). These
enzymatic reactions are also candidate systems for the
formation of ultimate carcinogenic metabolites in extrahepatic target tissues where sulfotransferase activity for Nhydroxy-2-acetylaminofluorene has not been detected (37,
87, 100). The acetyltransferase may be of special impor
tance in view of the wide range of tissues in which this
activity occurs and the very high reactivity of the product,
W-acetoxy-2-aminofluorene (6, 99, 100).
Both acetylated and nonacetylated aminofluorene adducts have been isolated from the livers of rats treated with
/V-hydroxy-2-acetylaminofluorene (5, 37, 107, 225) (Chart
12). On the basis of the products formed in in vitro reac
tions, the acetylated adducts must be formed primarily from
esters of /V-hydroxy-2-acetylaminofluorene, while the non
acetylated adducts are presumably derived either from
esters of A/-hydroxy-2-aminofluorene or from reaction of the
glucuronide of /V-hydroxy-2-acetylaminofluorene,
which
yields a mixture of acetylated and nonacetylated adducts
(130). While the methionine adducts (as evidenced by the
amounts of o-methylmercapto-2-acetylaminofluorene
and
o-methylmercapto-2-aminofluorene
isolated after degrada
tion of the liver proteins) comprise only about 10% of the
protein-bound fluorene derivatives (5, 37), the guanine
adducts that have been identified appear to account for the
major share of the nucleic acid adducts formed in rat liver
in vivo. The major adducts are those in which the substitu
tion occurs at C-8 of guanine (107), and the latter adducts
are much more readily removed in vivo from the DMA of rat
liver than is the minor adduci in which the substitution
occurs on the 2-amino group of guanine (106, 225).
JUNE 1978
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E. C. Miller
NUCLEIC
ions formed on protonation of N-hydroxy-2-naphthylamine
and N-hydroxy-4-aminobiphenyl
may be ultimate carcino
gens for the induction of urinary bladder tumors in the dog
and human (Chart 13). Thus, Kadlubar in our laboratory (94)
showed that these hydroxylamines
are N-glucuronidated
by
ACID ADOUCIS
the hepatic endoplasmic reticulum from these species, and
Radomski ef al. (170) have recently characterized
the Nglucuronide
of /V-hydroxy-4-aminobiphenyl
as a urinary
metabolite of 4-aminobiphenyl
in the dog. Furthermore, the
PROTEIN
ADDUCTS
PROTEIN
urines of many dogs and humans are sufficiently acidic to
hydrolyze the /V-glucuronides and to protonate the resulting
hydroxylamines
for reaction with DNA (94). These conclu
sions are consistent with the earlier report by Radomski
and Brill (169) on the quantitative correlation between the
carcinogenicities
of 1- and 2-naphthylamine
and 4-amino
biphenyl in the dog urinary bladder with the level of excre
tion of the corresponding
A/-hydroxylamines (plus the nitro-
Chart 12. Nucleic acid- and protein-bound adducts that have been identi
fied in the livers of rats treated with 2-acetylaminofluorene or N-hydroxy-2acetylaminofluorene.
An important
substitution of
carcinogenesis
the molecules.
question to be answered in considering the
macromolecules
by chemicals in relation to
is how the alterations affect the activities of
Fuchs ef al. (56) and Weinstein and Grun-
berger (220) have both considered this problem for DNA
substituted with 2-acetylaminofluorene
residues at C-8 of
guanine, and they have interpreted their structural findings
with similar models in which the guanine of the adduci is
rotated out of the double helix and the fluorene moiety is
inserted into the helix. These models are consistent with
base-pair substitution,
frame shift, and deletion mutations,
all of which have been observed with derivatives of 2acetylaminofluorene
(46, 120, 127). In addition, this substi
tution causes premature termination of transcription
(144)
and interferes with codon recognition of tRNA's (220).
Other Aromatic Amines and Nitro Compounds. The ac
tivities of all carcinogenic
aromatic amines, amides, and
nitro compounds appear to depend on their conversion to
N-hydroxy derivatives in vivo (30, 140, 141). The ultimate
carcinogenic metabolites have not been elucidated in most
cases, and the activation reactions may differ with the aryl
substituents, tissues, and species. For instance, /V-methyl4-aminoazobenzene
is activated similarly to 2-acetylamino
fluorene by /V-hydroxylation
and sulfonation
of the /V-hydroxy derivative, and the major nucleic acid adducts involve
substitution
of C-8 of guanine residues by the nitrogen
atom of /V-methyl-4-aminoazobenzene
(92, 93, 114). How
ever, while the oxidation of 2-acetylaminofluorene
is cata
lyzed by a cytochrome P-450 system, the /V-oxidation of the
dye is catalyzed by a flavoprotein
that does not require
cytochrome
P-450, and the data indicate that different
hepatic sulfotransferases
may act on the two substrates.
The carcinogen 4-nitroquinoline
1-oxide is reduced to 4hydroxyaminoquinoline
1-oxide, and Tada and Tada (204)
have shown that this hydroxylamine
can be esterified by
seryl-tRNA, The resulting seryl ester, which has been sug
gested as an ultimate carcinogenic
metabolite, reacts pri
marily with guanine and, to a lesser extent, with adenine
residues.
Recent studies have provided evidence that the nitrenium
1486
scarenes). Furthermore, bladder carcinomas were induced
in dogs by the instillation
of N-hydroxy-2-naphthylamine
but not 2-naphthylamine.
Polycyclic Aromatic Hydrocarbons. Studies on the me
tabolism of the polycyclic aromatic hydrocarbons were first
reported
in the late 1930's, and by 1950 Berenblum,
Schoental, Weigert, Mottram, Dobriner, and their associ
ates had observed phenolic and quinone derivatives of
several polycyclic hydrocarbons
in tissue preparations or
excreta of animals treated with these compounds (reviewed
in Ref. 29, Chap. 7). The knowledge of the sites and extents
of this metabolism was greatly extended during the next
two decades, especially by reports from the laboratories of
Boyland and Sims and of Heidelberger (reviewed in Refs.
40, 74, and 187).
* NADPH endoplasmic
+ 02
reticulum
V
endoplasmic
reticulumAr
A M--OH
,
H
LIVERti/H OH
VN^ArH
,UDPGA
|1
OHTRANSPORTH/—\
1,.
N'°HHoTTHArÕTÕH
PH<7Ar
V0"
<¡
N,H
URINEVH
Ar-IS1A
pH>7\^¿
'
^H-O
VH¿
/•
X^.
>H
*H®,-H,01
Ar M®
URINARYl
H
BLADDER
EPITHELIUMCOVALENT
¡
1'
¡METABOLIC
1 ACTIVATION
VREACTIVE
ELECTROPHILES
(ESTERS')
( FREE
RADICALS
>
~>)COW
BINDING TO
NUCLEOPHILIC
SITES
IN
CRITICAL
MACROMOLECULES
TUMOR
FORMATION
Chart 13. Formation and transport of possible proximate and ultimate
carcinogenic metabolites of arylamines for the induction of urinary bladder
cancer.
, possible transport or reaction. Ar, aryl substituent; UDPGA,
uridine diphosphoglucuronic acid (from Ref. 94).
CANCER
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VOL.
38
Current Perspectives on Chemical Carcinogenesis
As early as 1950 Boyland (19) suggested that the series of
metabolic phenols and dihydrodiols might be secondary
products of metabolically formed epoxides and that these
epoxides might be intermediates in tumor induction. At that
time the K-regions of the hydrocarbons (i.e., the phenanthrene-like double bonds) had been singled out by the
Pullmans (166) from calculations of electron densities as
likely critical sites for the interactions of the hydrocarbons
with tissue constituents, and the K-region epoxides were
therefore the first epoxides to be examined for carcinogenic
activity. The low carcinogenicities of the K-region epoxides
of benz(a)anthracene or related hydrocarbons on s.c. injec
tion or topical application to rats or mice in our laboratory
and those of Boyland, Sims, and Van Duuren were disap
pointing (21, 134, 186, 214). However, studies in Heidelberger's laboratory in 1971 and 1972 showed that usually, but
not always, the K-region epoxides were more active than
were the parent hydrocarbons for the transformation of
mouse fibroblasts in culture (62, 80).
In 1971 Selkirk ef al. (184) and Grover ef al. (61) showed
the formation by liver microsomes of unidentified epoxides
from benz(a)anthracene and dibenz(a,/?)anthracene. Work
from these laboratories also showed the electrophilic reac
tivity of the K-region epoxides (108). Knowledge of the
mechanisms of metabolic activation of the polycyclic
hydrocarbons and of the possible roles of the metabolic
products in carcinogenesis has since developed rapidly,
especially in the laboratories of Brookes, Conney, Gelboin,
Harvey, Jerina, Sims and Grover, and Weinstein. In 1974
Sims and his colleagues (188) expanded on an observation
of Borgen ef al. (14) that indicated that the critical metabo
lism of the polycyclic aromatic hydrocarbons might occur
at sites other than the K-regions; much evidence for this
concept has been presented since.
In the past few years particular attention has been fo
cused on the nature of the ultimate carcinogenic metabo
lites of benzo(a)pyrene and on the identities of its nucleic
acid-bound derivatives (Chart 14). Elegant studies from the
above laboratories now implicate 7/3,8a-dihydroxy-9a,10aepoxy-7,8,9,10-tetrahydrobenzo(a)pyrene
as a major ulti
mate electrophilic, mutagenic, and carcinogenic metabolite
of benzo(a)pyrene (81, 90, 95, 113, 221). As reported from
10
DNA
ULTIMATE
CARCINOGEN
Chart 14. The major route for the metabolic activation of benzo(a)pyrene
and the major adduci formed on reaction of the diol-epoxide with nucleic
acids. E.R., endoplasmic reticulum.
three laboratories, the major reaction products of this diolepoxide and its 9/3, 10/3 isomer with polyguanylic acid or
nucleic acids involve the 2-amino group of guanine resi
dues and C-10 of the epoxide (105, 156, 221). The synthetic
adducts are chromatographically identical with products
obtained on degradation of the nucleic acids from bronchial
expiants that had been incubated with [3H]benzo(a)pyrene
(221). The diol-epoxide also reacts in vitro, but to a much
smaller extent, with the cytosine and adenine residues of
polynucleotides (129, 222).
The analogous diol-epoxide derivative of benz(a)anthracene was suggested as an ultimate carcinogenic
metabolite of that hydrocarbon by Swaisland ef al. (199).
However, data from the laboratories of Conney and Jerina
now strongly indicate that the bay region or 1,2-position of
the angular ring of benz(a)anthracene may be analogous to
the 9,10-position of benzo(a)pyrene in the formation of an
ultimate carcinogenic diol-epoxide. Thus, the 3,4-dihydrodiol of benz(a)anthracene is considerably more carcino
genic for mouse skin than is benz(a)anthracene or any of
the other vicinal dihydrodiols (234), and the 1,2-epoxide
formed from the 3,4-dihydrodiol of benz(a(anthracene is a
much more potent mutagen without tissue activation than
are the two isomerie diol-epoxides with the substituents in
the 8, 9, 10, and 11 positions (233). The angular ring has
also been implicated as a critical site for the metabolic
activation of 7-methylbenz(a(anthracene (207) and of 7,12dimethylbenz(a)anthracene (149).
Unfortunately, it is impossible to give adequate recogni
tion in this brief review to all of the extensive work on the
mechanisms by which the polycyclic aromatic hydrocar
bons exert their carcinogenic activities. These studies have
given important new insights into hydrocarbon carcinogen
esis as well as further demonstrating the complexities of
the metabolic activations. Nevertheless, the knowledge in
this area is far from complete. Just as studies on the
alkylation of DNA by simple alkylating agents have shown
that the major adducts are not necessarily the ones that are
the most important in carcinogenesis (112, 189, 190), the
hydrocarbon-nucleic acid derivatives that have been identi
fied to date may or may not be those adducts most critical
for the initiation of carcinogenesis. Furthermore, since the
polycyclic hydrocarbons are promoting agents as well as
initiators, attention should also be given to the possible
promoting activities of the metabolites. Some of the meta
bolic phenols may be candidates for this role in view of the
promoting activities of a number of phenols (213).
Aflatoxin B,. Several observations led to the suggestion
that aflatoxin B, 2,3-oxide was the most likely ultimate
carcinogenic and reactive metabolite of aflatoxin B,. These
included the requirement of the 2,3-double bond for strong
carcinogenic activity (232) and the conversion by a mixedfunction oxidase system of aflatoxin B, (but not its 2,3dihydro derivative) to a toxic, mutagenic, and nucleic acidbinding derivative (58, 127). More conclusive was the
release by acid hydrolysis of 2,3-dihydro-2,3-dihydroxyaflatoxin B, from nucleic acids isolated from the livers of
aflatoxin B,-treated rats or from incubations of aflatoxin B,
and nucleic acids with fortified liver microsomes (201, 202).
Finally, the strong electrophile aflatoxin B, 2,3-dichloride,
synthesized as an analog of the 2,3-oxide which has thus
JUNE 1978
1487
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Research.
E. C. Miller
far eluded isolation from chemical or metabolic reactions,
was a strong carcinogen at sites of application (e.g.,
mouse skin and rat s.c. tissue) and a powerful mutagen
(203).
In the past year an acid degradation product of the major
adducts formed on reaction of the epoxide with nucleic
acids in microsomal systems in vitro has been independ
ently characterized as 2,3-dihydro-2-(guan-7-yl)-3-hydroxyaflatoxin B, in our laboratory (115), by Essigmann ef al.
(45), and by Martin and Garner (124) (Chart 15). In addition,
our data (115) and those of Essigmann ef al. (45) have
identified the same adduci as a major degradation product
of the nucleic acids from the livers of rats treated with
aflatoxin B,. While the overall yield of nucleic acid-bound
aflatoxin derivatives appears to correlate with the likelihood
of tumor formation (200), there are as yet no data that
specifically associate the aflatoxin B,-guan-7-yl nucleic acid
adducts with carcinogenesis.
Possible Molecular Mechanisms
esis
of Chemical Carcinogen
The above examples, while far from exhausting the liter
ature on metabolic activation, are sufficient to emphasize
that metabolic activation is an essential step in the induc
tion of neoplasia by most chemical carcinogens. The electrophilic ultimate carcinogens can react, probably more or
less indiscriminately, with a number of nucleophilic sites in
DNA's, RNA's, and proteins. Thus, the strong electrophilic
nature of ultimate carcinogens is consistent with both
genetic and epigenetic mechanisms of carcinogenesis or
with mechanisms that include both genetic and epigenetic
o rot liver
b. liver microsomes
+ NADPH»02
AFLATOXIN B,
AFB,-2,3-OXÅ’
ft\DECOMR
\PROOUCTS
|OFI
[ ]= presumptive structures
Chart 15. The metabolic epoxidation of aflatoxin B, (AFB¡),the major
reaction product of the epoxide with nucleic acids, and the degradation of
the nucleic acid adducts to yield the major product 2-(guan-7-yl)-3-hydroxyaflatoxin B, (///). The routes of formation of several other intermediates,
especially 2,3-dihydro-2,3-dihydroxyaflatoxin B, (//), on degradation of the
nucleic acid adducts are also shown. DECOMP., decomposition (from Ref.
115).
1488
events. These mechanisms may or may not involve the
expression of oncogenic viral information (see, e.g., Refs.
154, 165, 171, and 175).
Epigenetic Mechanisms. A fundamental basis for pro
posed epigenetic origins of cancer is the development of
complex organisms from single fertilized ova. During early
life each multicellular organism has many kinds of commit
ted cells that divide repeatedly to give rise to additional
cells with the same commitments. If, as is generally ac
cepted, these normal differentiations are the consequence
of epigenetic phenomena, similar epigenetic modifications
of cellular transcription or translation or both may also be
involved in the conversion of apparently normal cells to
tumor cells with relatively stable phenotypes (63). Further,
the now classic studies of Jacob and Monod (89) on the
circuits by which genetic expression in bacteria can be
more or less permanently altered, as well as the exquisite
controls for the expression or repression of information in
bacterial genomes (176), provide models for the induction
of tumors by chemicals through proliferation of cell lines
with altered transcriptional controls (163).
Data from a variety of experiments indicate that malignant
cells and nonmalignant cells may possess the same ge
nomes, although detailed analyses at the molecular level
have not yet been feasible. Gurdon (63) and McKinnell ef al.
(128) transplanted nuclei from frog renal carcinomas into
enucleated fertilized frog eggs with the subsequent devel
opment of apparently normal swimming tadpoles. These
studies appeared to show that the nuclei from the tumors
retained in expressible form at least the major share of the
information that was present in the fertilized ova. The
potential for the differentiation of malignant cells to nonmalignant cells was demonstrated by Pierce and his asso
ciates (161) for several types of tumors, including cloned
teratocarcinoma cells and stem cells from a transplantable
squamous cell carcinoma. Braun (22) has reported similar
differentiation of plant teratomas. Thus, cultures of teratomas could be grafted onto a plant where, under some
conditions, the progeny of the teratoma cells gave rise to
morphologically normal stems, leaves, and flowers, al
though the inherent neoplastic potential of the cells was
again evident on growth in culture (23). On the other hand
meiosis apparently caused the loss of the plasmid that is
essential for the malignant phenotype, and cells that devel
oped from the seeds grew as normal cells in culture (211).
Illmensee and Mintz (85, 145) have recently obtained chimeric mice by implantation of single cells from embryoid
bodies of mouse teratocarcinoma cells into mouse blástu
las. These chimeric mice contained a wide variety of appar
ently normal somatic tissues that developed from progeny
of the teratocarcinoma cells; in at least one case the
genotype of the teratocarcinoma cell was transferred to the
second generation through the sperm. Further studies on
the significance of these results in relation to the mecha
nisms involved in carcinogenesis are awaited with great
interest.
Genetic Mechanisms. In contrast to the epigenetic hy
potheses, which have as their fundamental premise that the
genomic information of tumor cells need not be altered
from that of normal cells of the same organism, the genetic
mechanisms assume that the change from normal to tumor
CANCER
RESEARCH
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VOL. 38
Current Perspectives
cell is dependent on genomic alterations. This latter point
of view receives primary support from the fact that the
potential of a cell is determined by the information coded in
the genome and from the abilities of all three types of
carcinogenic agents, viruses, radiations, and chemicals, to
alter cellular genomic content. Such genomic changes in
cells would be expected to occur most frequently as a result
of direct modification of DMA by the carcinogen. However,
modification of an RNA that was transcribed and integrated
into DMA or changes in the structure of a DMA polymerase
that resulted in a more error-prone enzyme could also lead
to altered cellular DNA's.
Auerbach, Demerec, and others were attracted in the
1940's to the idea that carcinogenesis
might involve mutagenie events and sought to correlate the carcinogenic
and
mutagenic activities of chemicals. Their data showed no
evident relationship
(reviewed in Ref. 26). However, the
situation changed markedly as the metabolism of chemical
carcinogens and the chemical nature of their active forms
became better understood. As we noted when we reviewed
this subject in 1971 (135), a qualitative correlation between
mutagenicity and carcinogenicity
was apparent when ulti
mate carcinogenic
forms were assayed in a nonmetabolizing system (transforming
DNA) or when nonultimate forms
were assayed in certain cellular systems (e.g., yeast, Drosophila) that possessed capacity for metabolism of foreign
chemicals. This correlation
has become better with the
supplementation
of bacterial mutagenicity
systems with
liver microsomes for the metabolic activation of carcino
gens (126, 127, 167, 197, 198). This correlation
between
mutagenic and carcinogenic
activities is a formal one and
is based on two facts: (a) that the ultimate forms of most, if
not all, chemical carcinogens are strong electrophilic
reactants; and (b) that, with the exception of the numerically
minor groups of the base analog mutagens and the simple
frame-shift mutagens that do not bind covalently, the ulti
mate forms of mutagenic chemicals are also strong electro
philic reactants. However, since these strong electrophilic
reactants also attack RNA's and proteins, this correlation
cannot be used alone to show that carcinogenesis
involves
mutagenic events.
Quite compelling evidence that tumor development may
depend on an alteration of genomic information is available
for UV-induced carcinogenesis.
Thus, xeroderma pigmentosum patients are very susceptible to the development of
skin cancer as a consequence of exposure to sunlight. As
shown by Cleaver, Bootsma, and others, the cells from
these patients have a greatly impaired capacity for the
error-free repair of DNA that contains UV-induced or certain
chemically induced lesions (reviewed in Ref. 32). Similarly,
as reported by Hart and Setlow (68), exposure of cells from
Poecilia formosa to UV/n vitro and subsequent inoculation
of the cells into new hosts gave rise to a high incidence of
thyroid tumors. The cells of this species contain a photoreactivating enzyme that cleaves pyrimidine dimers, and the
exposure of the irradiated cells to visible light after the UV
radiation largely prevented the development of tumors on
implantation of the cells.
Support for genetic mechanisms of transformation
in cell
culture is provided by the formation of stable, but revertible,
temperature-sensitive
malignant transformants of BHK cells
on Chemical
Carcinogenesis
by treatment with /V-nitrosomethylurea
or 4-nitroquinoline
1-oxide (15). Furthermore, recent studies by Marquardt ef
al. (122) suggest that Adriamycin, an intercalating drug and
frame-shift mutagen, may induce malignant transformation
in cell culture in the absence of detectable covalent inter
action with cell constituents.
Modern chromosome banding procedures provide some
evidence for associations of specific chromosomal
altera
tions with certain human cancers (152). Likewise, genetic
predispositions
for the development of some human can
cers can be interpreted as pointing to the involvement of
specific genetic components (2, 104). However, the inter
pretation of the latter data in a mechanistic sense is fraught
with problems, since genetic information
also determines
the metabolism of chemical carcinogens, the synthesis and
metabolism of hormones, and a variety of other factors that
may affect tumor incidence without being directly involved
in the initiation of cancer cells.
Overall, alteration of cellular DNA is currently viewed by
many and probably most investigators as the most attractive
mechanism for the initiation of carcinogenic
processes by
chemicals. Working on this premise an important question
is the nature of the DNA alterations that may be involved.
Some years ago Loveless (116) showed that the extent of
O6-methylation or ethylation of guanine residues in DNA,
reactions that lead to base substitution
mutations, corre
lated much better with the mutagenic activity of an alkylating agent than did the quantitatively
more prominent N-7
alkylation of guanine residues. Studies by Lawley and
others (112, 157) suggested a similar correlation
of O6methylation and ethylation of guanine residues in DNA with
the likelihood of tumor initiation by a series of methylating
and ethylating agents, but exceptions were evident. This
approach was refined in 1974 by Goth and Rajewsky (59),
who found that the persistence of the O6-ethyl guanine
residues in DNA, in addition to the amount originally
formed, appeared to be a critical factor for the induction of
tumors of the nervous system in rats. Further support for
the role of persistent O6-methyl or -ethyl guanine residues
in DNA have been obtained with other carcinogenesis
systems, although some apparent exceptions
have also
been reported (157). Furthermore, as emphasized recently
by Singer (190), it is too early to conclude that O6-alkylations of guanine are the most critical reactions, even for the
induction of mutations, since alkylations at this site may be
indicators of more critical alkylations at other sites, such as
the oxygen atoms of the pyrimidine bases in the DNA. To
the extent that chemical carcinogenesis
results from at
tacks on DNA, the great strides that have been made in
recent years in determining
the mechanisms
by which
specific types of damage to DNA result in mutations (32,
210, 230) will be of key importance in elucidation
of the
mechanisms of carcinogenesis
by chemicals.
Promotion of Initiated Cells. New observations are also
providing
insight into the possible molecular
bases of
promotion.
The phorbol esters, which have remarkable
activity in eliciting the development
of gross epidermal
tumors after application
of a small initiating
dose of a
chemical carcinogen to mouse skin, cause a progression of
changes. Application
of these promoters
results in in
creased synthesis of phospholipids,
RNA, protein, and DNA
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E. C. Miller
and an increased mitotic rate (17), but the earliest changes
appear to be increases in protease activity (209) and marked
increases in ornithine decarboxylase activity (18). The inhi
bition of the promotion stage of epidermal carcinogenesis
by protease inhibitors suggests that specific proteases may
be of critical importance in promotion (125, 209); increased
levels of one protease, plasminogen activator, frequently
accompany oncogenic transformation in cell culture (174).
Similarly, an impressive series of experiments show a close
correlation between the levels of induced ornithine decar
boxylase activity and promoting activity in mouse epider
mis. The latter correlation was obtained by comparison of
the effects of various doses and structures of promoters
(155) as well as by comparisons of the extent of inhibition
of tumor promotion and of ornithine decarboxylase induc
tion by a series of retinoid derivatives (217). The inductions
of both protease activity and ornithine decarboxylase activ
ity may have a common focus through their effects on the
levels or intracellular localizations of polyamines or pro
teins that modulate the expression of the DMA genome. The
epigenetic concept of promotion is in accord with its
relatively slow and reversible nature.
Extrapolation of Basic Knowledge of Chemical
genesis to the Prevention of Human Cancer
Carcino
Prevention of Initiation. Our still incomplete knowledge
of chemical carcinogenesis is already being applied to the
problem of reducing the future incidences of human can
cers. Thus, the generalization that strong electrophilic
reactivity is a basic requirement for ultimate chemical
carcinogens and detailed knowledge of enzyme systems
involved in the metabolic activation and deactivation of
many chemicals are providing useful tools for the recogni
tion of chemicals to be suspected of being potential carcin
ogens. Reasonable predictions can often be made of the
electrophilic reactivity of a chemical or its possible metab
olites from an inspection of its structure. Furthermore,
mutation systems, frequently fortified with metabolic acti
vation systems, are being used to screen for compounds
with potential electrophilic reactivity. The most common of
these mutation systems utilizes the Salmonella typhimurium
tester strains devised by Ames and his associates (1), but a
wide variety of other bacteria, fungi, plants, insects, and
mammalian cells are also being used (46, 79,167,197). The
mutagenic activities of chemicals in the mammalian tissuemediated bacterial systems have shown relatively good
qualitative correspondence with their carcinogenic activi
ties for experimental animals and, where known, for hu
mans (126,127,135,167,197,198).
These assays, however,
give some false-positive and some false-negative results;
i.e., they have failed to show mutagenic activity for about
10% of established carcinogens, especially some of the
weaker ones, and they have shown mutagenic activity for
some chemicals that have thus far not induced tumors in
animal tests.
Assays have also been developed in which the end point
is the malignant transformation of mammalian cells in
culture, and approaches to the fortification of these assay
systems with metabolic activation systems are being stud
ied. The transformation assays are currently being evalu
1490
ated for their abilities to predict the carcinogenic potentials
of chemicals, and preliminary results are encouraging (39,
54, 160, 177). However, up to the present, and for the
foreseeable future, none of these mutagenicity or transfor
mation prescreens appears tojpe reliable enough to replace
whole animal carcinogenicity assays for the evaluation of
carcinogenic activity.
Other approaches to the reduction of contact with ulti
mate carcinogens involve alteration of carcinogen metabo
lism so that less is converted to ultimate forms or enhance
ment of the intracellular levels of nucleophilic acceptors
that can react in noncritical ways with the electrophilic
ultimate carcinogens. Experimental data have been col
lected for both of these approaches, and, as examined
especially by Wattenberg (218), marked protection has been
achieved in a number of model systems in experimental
animals. At present, it is difficult to explore these ap
proaches in the human in view of the uncertainties of the
ramifications of such manipulations. Possible untoward
effects include an increased metabolic activation of some
carcinogens or increases in other toxic reactions (229).
Further, some chemicals, such as phénobarbital,that have
inhibited tumor induction when administered simultane
ously with a carcinogen, have shown promoting activity
when applied subsequent to an initiating dose (158, 159).
Reduction of human hazard also requires knowledge of
where, how, and to what extent carcinogens are present in
particular places. We are surprised all too often by our lack
of foresight. Thus, in 1950, many years after the carcino
genicity of 2-naphthylamine for the human bladder and its
hazard in industrial situations was generally recognized,
Case and Hosker (28) traced a cluster of cancers of the
urinary bladder among rubber workers to 2-naphthylamine
which was present as an impurity in the antioxidant then
used for curing rubber.
Study of the large group of carcinogenic W-nitroso com
pounds has provided similar evidence of the need for acuity
in looking for potential exposures (147). An early concern
was the likelihood of formation of nitrosamines or nitrosamides in the stomach after ingestion of amines or amides in
the diet or as drugs together with nitrite (13,119). However,
Tannenbaum, Preussmann, and their associates (193, 205)
have now shown that the main source of stomach nitrite is
dietary nitrate which is a normal constituent of many foods,
especially certain vegetables. After absorption the nitrate is
excreted in the saliva and reduced by the bacteria in the
mouth; the nitrite thus formed reaches the stomach through
the normal swallowing of saliva.
A/-Nitroso compounds have also been found as pollut
ants, fortunately usually at relatively low levels, in urban air,
water, soil, and a variety of commercial products (13). For
instance, Fan and his associates (47) have recently reported
concentrations of 0.02 to 3% of A/-nitrosodiethanolamine,
which induces liver tumors in the rat, in synthetic cutting
oils that contain triethanolamine and were placed in metal
cans treated with nitrite to prevent corrosion. These syn
thetic cutting oils were introduced some years ago as a
replacement for the mineral oil-based products that had
posed a risk of skin cancer to workmen. While the impact
of these exposures on human cancer incidences is diffi
cult to evaluate, it is apparent that we need much better
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VOL. 38
Current Perspectives
communication
between those who formulate products and
their packaging, those who have good knowledge of the
adverse biological effects of chemicals, and those who can
predict the reactions that can be anticipated
in given
mixtures of chemicals under a variety of conditions.
Fur
thermore, astute clinical observations and careful epidemiological studies will continue to be important approaches
for assessing the success of our predictive and preventive
approaches and for recognizing those situations that re
quire further study.
Prevention of Promotion of Initiated Cells. Little work
has been directed toward the detection of promoting agents
that may cause initiated cells to develop into gross tumors,
although such chemicals must also occur in the human
environment. A very important approach to the modification
of promotion and reduction of cancer incidences appears
to be available through administration
of certain retinoids.
Vitamin A deficiency
has long been known to result in
hyperplasia and keratinization
of epithelia, and Lasnitzski
(111) some 20 years ago noted that high levels of vitamin A
prevented and even reversed the hyperplastic effects in
duced by 3-methylcholanthrene
in organ cultures of mouse
prostates. Building on these and other results, Sporn,
Bollag, and others (see Ref. 194) envisioned the synthesis
of retinoids that can maintain differentiated epithelia in the
face of carcinogenic
insults, that lack the toxicity of high
levels of naturally occurring vitamin A, and that are retained
by extrahepatic tissues. The developments of the past few
years are very promising. Reductions of the incidences of
cancers of the skin, lungs, urinary bladder, and breast in
experimental
animals have been obtained even when the
administration
of the retinoid was not begun until after the
treatment with the chemical carcinogen
had been com
pleted (194). Thus, through maintaining the differentiation
of the epithelia previously exposed to the initiating activity
of a chemical carcinogen, the retinoids are apparently able
to prevent some promoting influence associated with the
hyperplastic state. The application of the synthetic retinoid«
to the inhibition
of carcinogenesis
in high-risk groups,
such as those known to have been exposed to urinary
bladder carcinogens, is receiving serious consideration.
Concluding Remarks
It is apparent that there is need for much further research
to unlock the doors to a complete understanding
of the
processes involved in the induction of cancer. Neverthe
less, a comparison of our state of knowledge today com
pared with that of 35, 25, or even 10 years ago encourages
me in the belief that further study will eventually bring a
detailed insight into the reactions involved in the initiation,
promotion, and progression of cells in the neoplastic proc
ess. This detailed knowledge will surely give us the best
foundation for the prevention and possibly the treatment of
human cancers.
In the meantime, it is clear that the tremendous effort that
has gone into the study of experimental chemical carcino
genesis and into the epidemiology
of human cancer over
the past 20 to 30 years has given us much information
relevant to the prevention of cancer. Thus, these studies
have pinpointed certain industrial carcinogens, have clearly
on Chemical
Carcinogenesis
indicated that each individual can markedly reduce his
likelihood of developing cancer by reducing his exposure
to sunlight and cigarette smoke, and have given us methods
for detecting potential human carcinogens. Much data have
clearly established
that chemical
carcinogenesis
is a
strongly
dose-dependent
phenomenon,
although
the
shapes of the dose-response curves are rarely determined
over wide ranges and probably cannot be established for
the low exposures of interest to human populations.
The
strong dose dependence
is too often slighted in public
discussions
of possible human hazards from mutagenic
and/or carcinogenic
chemicals. While specific chemicals
are known to pose risks to certain human populations at
relatively high levels of exposure and others are suspected
of posing such risks, wide-spread prohibition of the use of
such chemicals at much lower levels may or may not be
desirable. Likewise, the extrapolations
of carcinogenicity
data for experimental animals and of mutagenicity data to
evaluations of possible hazards for human populations are
very difficult problems. The single fact that, at very high
doses, a chemical causes some cancers in experimental
animals or some mutations may not be an adequate reason
for removing it from uses beneficial to the public." The
questions to be asked must include how much risk a given
low-level use may entail and how much benefit would be
lost to society from restriction on the use of that chemical
for that purpose.
It is important that we recognize that life is a series of
risks and benefits. I have been much impressed with the
writings by W. W. Lowrance on safety matters. Lowrance
(117) defines safety "as a judgement of the acceptability of
risk." Risk, in turn, he defines "as a measure of the
probability
and severity of harm to human health." He
concludes that a "thing is safe if its attendant risks are
judged to be acceptable."
How to decide what acceptable
levels of risk are for each individual and for the population
as a whole are important
societal problems that must
receive much more attention. In highly industrialized
soci
eties the activities of each of us have some impact on other
members of the society. At the same time rigid restrictions
of scientific,
personal, societal, -and industrial
activity,
where they are not really needed for the common good, can
easily lead to important losses to society.
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CANCER
RESEARCH
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VOL. 38
Some Current Perspectives on Chemical Carcinogenesis in
Humans and Experimental Animals: Presidential Address
Elizabeth C. Miller
Cancer Res 1978;38:1479-1496.
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