Download Chemical carcinogenesis: a current biological

Cardnogenesb Vol.5 no.l pp.1 - 5 , 1984
Commentary
Chemical carcinogenesis: a current biological perspective
Emmanuel Farber
Departments of Pathology and Biochemistry, University of Toronto,
Banting Institute, 100 College Street, Toronto, Ontario, M5G 1L5, Canada
cell populations is a common feature. This again makes any
delineation of a late discrete fine end-point difficult if not impossible.
Introduction
The ultimate end-point of a cancer-inducing exposure to a
carcinogen is the appearance of a localized collection of cells
(i) undergoing inappropriate proliferation, (ii) expanding
locally and invading the surrounding tissue and (iii) releasing
cells that generate in turn new collections of proliferating cells
in distant sites. Most cancers are made up of heterogeneous
cell populations with considerable diversity in cell structure,
in chromosomal and genetic makeup and in biochemical properties as well as in states of differentiation. Although the cell
type and biological behaviour do vary with different carcinogens, there is no known relationship between the nature and
metabolism of the carcinogen and the nature of the ultimate
cancer. Even though a minority of chemical carcinogens induce highly predictable types of neoplasms, e.g., vinyl chloride, 1,2-dimethylhydrazine, /3-naphthylamine, etc., modification of the conditions of exposure can change the spectrum
of cancers induced. To cite but one example: with
1,2-dimethylhydrazine and some other carcinogens, acute exposure, when coupled with surgical partial hepatectomy, may
induce a high incidence of liver cell cancer while in the
absence of the surgical intervention, few or no such
neoplasms are seen. To date, major correlations exist only
between the cell or tissue distribution of the metabolizing
and/or repair enzymes and the tissue site of cancer development but not with the nature of the cancer.
Thus, in most instances, cancer as an end-point and the
types of cancer give little insight into how the chemical might
be carcinogenic. Also, despite the intensity of human and
material resources that have been focussed on cancer per se,
the essential biochemical and genetic basis for the different
major properties of cancers, such as autonomy of growth, invasion and metastasis, continue to elude the cancer researchers.
It is apparent, however, that the major behavioural properties of cancers are not acquired as a single step but rather seriatim or sequentially. This also pertains in most instances to
the steps preceding the appearance of any of these characteristics. When and where studied, chemical carcinogens are related to cancer through a stepwise process that may often involve one half or more of the normal life span of that species.
For example, in some rodents, a carcinogenic process lasting
one or even two years is not uncommon. In humans, 20 years
or longer is not unusual for many types of cancer. Also, once
a collection of obviously malignant neoplastic cells does appear relatively late in the process, this is by no means a fixed
end-point. Cancers in general continue to undergo further
progression with selection toward increasing autonomy and
invasion. Thus, in carcinogenesis, we are dealing with flexible
systems of cellular changes in which cellular evolution to new
The carcinogenic process
Given this state of the art, which unfortunately has not
changed radically in several decades, increasing emphasis
must be placed upon the pathogenesis of cancer, i.e., how
does cancer develop, if we are ever to understand the
mechanisms of how chemical carcinogens, both initiating and
promoting, relate to cancer.
The past 15 years or so have seen a phenomenal development of knowledge concerning the metabolism of carcinogens, the nature of their activated forms, the interactions of
such reactive derivatives with DNA and other cellular constituents and the repair systems for carcinogen—DNA adducts. Deeper insights have been generated into how these
various components may be modulated by important endogenous and exogenous influences such as hormones, genetic
background, nutrition and chemicals.
During this same period, some elementary principles of
chemical carcinogenesis in the skin of mice and rabbits,
especially the concept of initiation and promotion, have been
extended to several other systems including the liver, urinary
bladder, pancreas, colon and lung (1—3). Models are now
available or are being developed that involve the ultimate appearance of cancer by two manipulations or operations, a
relatively brief initiation and a more prolonged imposition of
a promoting influence.
When viewed from the point of view of cell response and
cell behaviour, i.e., from a biologial perspective, what is
emerging from these various studies is a fairly clear-cut picture of the panorama — the dominance of certain patterns of
tissue response and some of the key qestions that should be
stressed.
Although we do not know all the various patterns of tissue
and cellular responses that lead to cancer, one pattern stands
out in many tissues with the vast majority of chemical carcinogens. In the skin, liver, colon (in some species), pancreas
and several other sites, dicsrete new focal collections of cells,
called nodules, papillomas or polyps, are virtually constant
accompaniments of the carcinogenic process during the long
precancerous or preneoplastic period. In the skin, in the liver
and in the colon, these focal collections have been shown to
be sites in which cancer can arise. Their possible role in the
development of cancer in some models has been clearly established.
These focal collections arise from tissues that have been exposed to carcinogens under conditions of initiation. The
nature of their progenitors in initiated tissues, so called
'initiated cells', their mode of development and their possible
role in carcinogenesis are considered to be crucial items in my
opinion and are one major topic for discussion. The second
item of major importance in any discussion of carcinogenesis
© IRL Press Ltd., Oxford, England.
1
E.Farber
concerns the sequence of changes that these focal proliferations undergo as part of their possible role in the genesis of
cancer. We consider these two topics to be central to the
scientific analysis of the carcinogenic process in the present
day perspective and their clarification to be essential to bridge
the large gap between the chemistry and biochemistry of carcinogens and cancer development.
Genesis of nodules, papillomas and similar lesions
From the point of view of cancer development, the first major step is the genesis of altered cells during initiation.
Although long suspected (4), it has only fairly recently been
established that initiation in the liver (5 — 8) and probably in
other quiescent organs (9,10) requires a round of cell proliferation. This may well explain the observations made many
times that metabolism with adduct formation in the liver with
many carcinogens does not initiate carcinogenesis, unless the
carcinogen induces cell death with regeneration (see 11)..
In the systems studied, and with the current assays, very
few altered cells seem to be generated during initiation. This
observation is consistent with the hypothesis that initiation is
a rare event that could be due to some very uncommon
change in the target cells, such as a mutation or some other
type of genetic alteration.
What is the nature of the alteration in initiated cells that
allows them to be expanded during promotion? The ability to
be expanded, so called 'clonal expansion', is the phenotypic
expression that dominates the phenomenon of promotion.
Attempts to understand this fundamental question are continuously frustrated by an underlying consideration. Since
any focal 'clonal' expansion involves a difference between the
cells to be expanded and the surrounding cells and tissues, it
becomes important to understand the biological nature of the
expansion before any attempts at molecular mechanistic
analysis are made. This may be best illustrated by an example. In any solid tissue, such as the liver, the presence of
nodules is a reflection of a difference in how the nodule
precursor cells respond as compared with their surrounding
cells. If the 'initiated' cells have acquired a resistance to inhibition of cell proliferation by carcinogens or other compounds while the surrounding cells have not, if the liver is exposed to an appropriate inhibitor or cell proliferation and if a
stimulus for cell proliferation is provided to the liver, the few
'initiated' resistant cells will respond 'normally' by proliferation while the majority will not. Thus, nodules can be rapidly
generated under these conditions, as occurs in the resistant
hepatocyte model of liver carcinogenesis (12). This rapid appearance of hepatocyte nodules promotes cancer development. In this case, the mechanistic analysis of promotion
must focus on at least two aspects of the system: (i) how
specific or general is the resistance and what is its biochemical
and genetic basis? and (ii) is the 'clonal expansion' sufficient
for promotion to cancer or does the promoting environment
impose some additional genetic or other change on the cell? If
the acquired resistance is not a property that can be used for
clonal expansion, its study as a basis for promotion would be
quite irrelevant to carcinogenesis.
It is also probable that this mechanism of 'clonal' expansion, which may be called 'differential inhibition', is not
universally applicable to the development of nodules and promotion in all models. For example, no differential inhibition
is seen with orotic acid promotion, yet this is an excellent promoter for liver cancer (13). Perhaps in this or other models,
the 'initiated' hepatocytes show a difference in their primary
response to a mitogenic stimulus, i.e., 'differential stimulation'. Clearly, such a biological basis for clonal expansion
and promotion requires a different type of mechanistic
analysis with an entirely different orientation.
This consideration also applies to the skin and all other
organs or tissues in which focal collections of proliferating
cells play a role in the carcinogenic process. How can one
hope to understand a biochemical or molecular mechanism of
a phenomenon, such as papilloma or nodule formation, without some understanding of the phenomenon under study?
This seems appropriate for any biochemical, molecular or
genetic analysis of a complex process, including those in
disease. It raises the important question: can we hope to uncover the basic biochemical mechanism of any chemicallyinduced phenomenon, but especially one with the appearance
of new cell populations, by only following the metabolism
and fate of the chemical? Certainly, from today's perspective,
a heavier emphasis on the reverse approach, the delineation
of the biological phenomenon and then its mechanistic
analysis, would seem to be equally or perhaps more appropriate.
In respect to the genesis of nodules, papillomas and
analogous lesions, do different promoting environments
select different types of altered cells in an initiated tissue or is
there only one type of 'initiated' cell? Although fundamental,
this question obviously must await a better understanding of
how different promoting environments in each tissue select
carcinogen-altered cells for expansion. Since initiated cells,
after expansion, show a whole pattern of phenotypic changes
and not just one or two, it is conceivable that different promoting environments use different 'handles' in the same initiated cell for their selections.
A common pattern of gene expression
One of the more striking aspects of chemical carcinogenesis
are the observations that the nodules, papillomas and polyps
generated in quite different models show an unusual degree
of similarity in cellular composition and organization and in
other properties. This has been most intensively studied in the
liver.
The hepatocyte nodules, generated in five different models
(intermittent chronic exposure, resistant hepatocyte, chronic
enzyme induction (phenobarbital), choline deficient diet,
orotic acid (see 14,15)) are in general similar not only in
respect to architectural arrangement of hepatocytes, blood
supply and negative and positive histochemical markers (14)
but also in their metabolic biochemical pattern (16 — 23). The
pattern can be related to an increased resistance to many
xenobiotics. The decreased metabolism of carcinogens by the
microsome mixed function oxygenase system (decrease in
cytochromes P450 and in several oxygenases), the increased
conjugation of reactive metabolites with glutathione and
glucuronic acid (increase in glutathione, glutathione-Stransferase and a UDP-glucuronyl transferase), the decrease
in sulfotransferase, the accelerated hydration of epoxides by
epoxide hydrolase, and the accelerated reduction of quinones
by DT-diaphorase (quinone reductase) present an unusually
special phenotypic program consistent with a general increase
in resistance to the cytotoxic effects of some foreign
materials. Studies in whole animals have shown that the biochemical pattern is reflected in altered pharmacodynamics of
at least one carcinogen, 2-acetylaminofluorene. Decreased ac-
Cbemkal cardnogenesis
tivation and more efficient conjugation and excretion in the
urine are among the obvious differences between control
animals and animals with nodules in their liver (24). Also,
with two carcinogens and two hepatotoxic chemicals,
decreased activation, decreased interaction with macromolecules and decreased acute cytotoxicity are seen in nodules
(25). Another interesting feature is the presence of a common
pattern of change in cytosolic proteins, the most obvious
change being a considerable increase in a 21 kD protein in
nodules generated in each of the five models (21).
This commonality in gene expression appears to be constitutive, since it is quite independent of the continuing exposure to the carcinogen or promoting environment in at least
some instances. For example, it is seen in nodules 15 months
after transfer to the spleens of syngeneic control rats. These
transfers often evolve into unequivocal hepatocellular carcinoma (26).
It should be pointed out that the biochemical or metabolic
patterns have not been explored in nodules generated by carcinogens that are not mutagenic or only mildly so or by the
feeding of choline deficient diets without added carcinogens
(27,28). The best examples are with the hypolipidemic agents
that induce nodules and hepatocellular carcinoma but are not
positive in many short term tests (29,30). Whether these
nodules will be similar to those seen in the other five models is
an interesting question.
Nodule or papilloma to cancer sequence
Options for nodules and papillomas
During initiation, tens (in the skin) or hundreds or thousands
(in the liver) of altered cells are often induced and each of
these can be expanded to form papillomas or nodules by an
appropriate promoting environment. Yet, in most models,
only one cancer or perhaps a few may be seen even under optimal conditions. This is also the experience in the colon in
some humans who have an hereditary disease called multiple
polyposis. Only very few of thousands of polyps act as
precursors for cancer development.
In at least the skin and the liver, the crucial turning point in
the carcinogenic sequence to cancer occurs at the nodule
stage. In both of these sites and probably also at other sites
such as the colon, the majority of the papillomas or nodules
in some models often 'disappear' as such and only a small
minority persist. However, there are indications that the
percentages of nodules or papillomas that remodel may vary
with the model. For example, in the skin, more papillomas
regress when the promoter is an active phorbol ester than
when it is a carcinogen (31).
The persistent nodules or papillomas in turn are the sites
for further focal change in a few cells to generate new cell
populations. In the skin, ultimately, cancer may appear in
such papillomas by a process as yet undescribed. In the liver,
the few persistent nodules are the sites for new focal cell
populations ('nodules in nodules') (15). This new population
may expand and in turn may become the site of a further
focal cellular change. How many such steps are involved in
cancer development is unknown. However, in a few instances, metastasizing cancer can be seen to arise in such a
manner.
A critical question for cancer development then becomes
the basic nature of the 'disappearing' process of the majority
of nodules or papillomas and the reasons for persistence. The
'disappearance of papillomas' in the skin has not been
studied very much. In the liver, recent work has clearly
established that the nodules 'disappear' by undergoing
remodeling to normal-appearing liver by a process of redifferentiation (32). For unknown reasons, the few persistent
nodules either fail to show the redifferentiation or do it very
slowly. Presumably, the new focal cell population arises in
such nodules before redifferentiation can take place. Unlike
the population of early nodules, the persistent ones grow well
in the spleen and may evolve into cancer (26,33).
An important question that remains unexplored to date is
whether the two major options elected by nodules or papillomas are determined by the exposure to the carcinogen during initiation or by the promoting environment or are
stochastic and based only on chance and probability.
It must be emphasized that once the persistent nodule is
generated, no further external manipulations or operations
are needed for cancer development. In other words, the
longest period in the carcinogenic process, the nodule-papilloma to cancer sequence, is 'self generating'. This period consists of steps, probably several, yet somehow it can progress
without the need for any other external manipulation by the
investigator. Is this compatible with a popular speculation on
the 'multi-hit' formulation? In two systems, the skin and the
liver, some further progression beyond the papilloma or the
nodule has been observed with the further exposure to carcinogens (34,35). Is this truly a reflection of what is happening during the long 'self generating' period in chemical carcinogenesis?
Subsequent steps
As already pointed out, malignant neoplasia is itself a stepwise process with at least three discrete phenomena. Judging
from our current knowledge about the liver models, there
would appear to be a further stepwise sequence between the
persistent nodule and the first appearance of a probable
malignant neoplastic population.
Overall, this period in the carcinogenic process appears to
be a long one. In at least some models, the overall rate
limiting step or steps reside somewhere in this sequence. Its
nature remains to be studied. However, it is already evident
that modulation of a major magnitude can exist in this
period. For example, in one model of liver carcinogenesis, the
administration of progesterone can significantly increase the
growth of a few nodules while (3-estradiol accelerates
remodeling (36). It is anticipated that diet, drugs and hormones might have a major impact upon the carcinogenic process through exercising some influence on the persistent
nodule-papilloma to cancer sequence.
This raises an important general feature. In the skin system, there is evidence that the genesis of papillomas may be
divisible into at least two steps and that each can be influenced by different agents (37,38). Clearly, a goal of research in
carcinogenesis is to divide the process into as many steps as
are real and then to begin to place them into a meaningful
perspective so as to understand their role in the development
of cancer.
Some general considerations
One of the most interesting questions concerning the
mechanisms in chemical carcinogenesis is: what are the contributions of a carcinogen to cancer development? As initiators, most carcinogens clearly induce a few altered cells in
the target tissue and these few cells can act as sites of cellular
expansion to form papillomas, nodules, polyps and similar
E.Farter
focal collections. Is this the sum total of the contributions of
the initiating component of a carcinogen?
As promoters, carcinogens also induce the expansion of the
few cells altered during initiation. Does the carcinogen add
any other information to the expanding cell population or only a means for 'clonal' expansion? Under these conditions, is
the 'self generating' nature of the subsequent progression to
cancer merely an illusion? Perhaps the promoting or initiating
action of the carcinogen also induces built in changes that require many cell cycles or merely time to express themselves?
Is this feasible, given the number of steps involved in the process until the stage of metastasizing cancer is reached?
In vitro versus in vivo
The emphasis in this presentation has been on in vivo. Since
an increasing number of studies are being done on in vitro
systems, what is their relevance to what happens in the intact
animals or in humans? The in vitro transformation with
chemicals occurs in a stepwise fashion (39). Also, promoters
as well as initiators seem to have analogous effects in vitro
and in vivo. However, the identification of new cells and their
nature remains to be delineated in a manner analogous to
what is being developed in vivo. Presumably, it may become
possible ultimately to superimpose at least some of the in
vitro steps on those in vivo, since detailed mechanistic studies
will require the availability of appropriate in vitro models for
the different steps.
Oncogenes and carcinogenesis
A topic of great current interest is the step or steps at which
some of the oncogenes fit into the biological perspective of
carcinogenesis (40). It is too early to make any judgements,
even very tentative ones. Since much of the recent work on
human cancers has involved very advanced cancer or even cell
cultures from such cancers and since only a maximum of
15% of any type of cancer has been positive in transfection
assays, it is impossible to place 'the oncogene' into a specific
site in the stepwise carcinogenic process today. However,
three recent reports (41—43) suggest that some oncogenes
may work in concert with each other or with viruses or
chemicals in human or rat cells in culture in a stepwise transformation in vitro. The effects of the oncogenes were manifested after the effects of the chemicals or viruses in a process
that involved at least two or three steps. Clearly, a major
challenge is now offered by the recent work on oncogenes. If
they play a role in the carcinogenic process and are not an expression of advanced progression in the post-cancer sequence,
at what step or stage do they participate and what is/are their
mechanism(s) of action?
Carcinogenesis as a physiological process
The common properties of the hepatic nodules, the ability of
nodules to undergo the very complex process of redifferentiation during remodeling and the nature of the changes seen in
the preneoplastic segment of carcinogenesis suggest that at
least some of the earlier steps are physiological, as opposed to
aberrant or 'pathological' and that they might represent a
form of adaptation to xenobiotics with survival value (3).
Based upon these and other considerations, it may be appropriate to begin to formulate new specific hypotheses for
initiation. The current experience in the biochemical-biological synthesis in hepatocarcinogenesis suggests the possible
role of a major regulatory gene for the constellation of
biochemical changes seen in expanded initiated cells. This
major regulatory gene would control in a coordinate way pat-
terns of gene expression relating to the handling of many
xenobiotics by the liver cells.
Judging by the experience to date in carcinogenesis in the
rat liver, it might be profitable to begin to view the process of
cancer development with chemicals in a somewhat broader
biological context than heretofore and to further explore the
close interplay between chemistry, biochemistry and biology.
Acknowledgements
We wish to thank Helene Robitaille for the valued secretarial assistance and
Drs. D.S.R.Sarma, M.A.Hayes and R.Lebovitz for many helpful suggestions. The author's research was supported by grants from the National
Cancer Institute of Canada, the Medical Research Council of Canada
(MT-5994) and the National Cancer Institute, National Institute of Health
(USA)(CA-21157).
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(Received on 29 August 1983; accepted on 26 September 1983)