Epigenetic Mechanisms of Chemical Carcinogenesis
James E. Klaunig. Ph. D.*, Lisa M. Kamendulis, and Yong Xu
Division of Toxicology, Department of Pharmacology and Toxicology,
Professor and Director of Toxicology, Indiana University School of Medicine, 635 Barnhill Drive, MS-1021, Indianapolis, IN 46202-5120,
Tel: (317) 274-7824
Division of Toxicology, Department of Pharmacology and Toxicology,
Linkage between exposure to chemicals and the induction of cancer in rodents has been well established. A cause- and-effect and dose-responsive relationship has been established between chemical carcinogen exposure and subsequent development of neoplasia. Investigators in the mid-twentieth century established that a multi-staged process was involved in cancer development. Subsequent work showed that chemical carcinogens can function at several of the stages of the tumorigenesis process. It has become apparent that chemical and physical agents that induce cancer may do so through different cellular and molecular mechanisms. Weisburger and Williams (1983), recognizing the apparent differences in the ways compounds participate in the carcinogenesis process, coined the phrases "genotoxic" and "epigenetic" in describing activities of chemicals and physical agents that induced cancer. The term "nongenotoxic" has to some extent replaced "epigenetic" and thus, classification of chemical carcinogens has been frequently delegated to either the genotoxic or nongenotoxic categories.
While the specific definitions of genotoxic and nongenotoxic will vary with the individual or organization using these labels, there are generally agreed upon attributes for placement in these categories (Table 1). Genotoxic agents usually refer to chemicals that either directly bind to or damage genomic DNA, which in turn can result in mutation. Agents that participate in this category are usually reactive compounds whose activation occurs in cells. Genotoxic agents produce a dose-response increase in neoplasm formation. Although within the dose response profiles of most genotoxic agents a threshold dose is apparent, for regulatory purposes, genotoxic carcinogens are assumed to be without threshold. Selective examples of genotoxic carcinogens are shown in Table 2. Most genotoxic agents are highly mutagenic in in vitro eukaryotic and prokaryotic systems. Frequently, these compounds invoke their tumorigenic response at or close to the site of application or administration of the compound. A second category includes genotoxic agents that require metabolism or activation by cellular metabolic pathways to produce their effect. These chemicals frequently invoke their effect in target organs that are able to metabolize and activate the procarcinogenic form of the chemical to its ultimate DNA interactive form. Based on this requirement for activation, procarcinogenic genotoxic chemicals usually are tissue-specific. As noted above, genotoxic agents eventually result in modification to cellular DNA. This DNA reactivity results in mutation in the target cell.
Table 1: Biological Characteristics for Classification of Genotoxic and Nongenotoxic Carcinogens
Direct DNA reactivity
Tumorigenicity is dose response
Can be complete carcinogens
Usually not strain or species specific
Functions at initiation and progression stages of
Nondirectly DNA reactive
Usually exhibits strain, species and tissue specificity
Functions at the tumor promotion stage of the cancer process
Table 2: Selected Examples of Genotoxic and Nongenotoxic Hepatocarcinogens
Selected Examples of Genotoxic Hepatocarcinogens
Nitrosamines (Diethylnitrosamine, Dimethylnitrosamine)
Polycyclic Aromatic Hydrocarbons
Mycotoxins (Aflatoxin B1)
Aromatic amines (2-AAF, 2- Napthylamine, 4 Aminobiphenyl)
Selected Examples of Nongenotoxic Hepatocarcinogens
Chlorinated compounds (Carbon tetrachloride, Chloroform)
Organochlorine pesticides (Dieldrin, DDT, Chlordane)
Peroxisome proliferators (DEHP, Clofibrate, Nafenopin)
Other organochlorine compounds (TCDD, PCBs)
Hormones (Estradiol, Diethylstilbestrol)
Barbiturates (Phenobarbital, Sodium barbital)
In recent years, a second category of carcinogenic compounds that appear to function through non DNA reactive mechanisms has been identified. These nongenotoxic or epigenetic agents do not induce mutation in short term eukaryotic and prokaryotic mutation assays nor induce direct DNA damage in the target organ. Several examples of these agents have been identified. In Table 2 selective hepatic nongenotoxic agents are listed. These agents modulate cell growth and cell death and exhibit dose response relationships between exposure and tumor formation. While the exact mechanism(s) of action of these agents on the process of neoplastic cell formation has not been established, changes in gene expression and cell growth parameters appear to be paramount in their mode of action. Nongenotoxic compounds exhibit temporal and threshold characteristics frequently requiring chronic treatment to produce carcinogenicity. Many nongenotoxic carcinogens appear to impact on the promotion stage of the cancer process.
The multistage nature of the carcinogenesis process has been well defined. Based on experimental evidence three distinct stages of the carcinogenesis process have been identified. These stages, initiation, promotion and progression are dissectable by biological and morphological characterization (Table 3). Initiation involves the formation of a preneoplastic cell resulting from the "fixing" of a mutation by a round of DNA synthesis. The process of initiation involves a genotoxic event that is irreversible resulting in the formation of the initiated cell. The promotion stage involves the selective clonal expansion of the initiated cell through an increase in cell growth or a decrease in apoptosis. The events of this stage are dose dependent and reversible upon removal of the tumor promotion stimulus. The third stage, progression, involves genotoxic events that result in changes from the preneoplastic to the neoplastic state. This stage is irreversible and involves changes in ploidy and chromosome integrity.
Table 3: Biological Characteristics of the Stages of Carcinogenesis
Genotoxic event (mutation)
Formation of preneoplastic cell
Exhibits dose response properties
Possible "spontaneous formation of initiated cell"
Cell division necessary to "fix" mutation
Apparent lack of threshold dose response
Changes in gene expression
Selective clonal expansion of preneoplastic cell population
Clonal expansion dependant on constant exposure to agent
Exhibits dose response properties
Karyotypic changes and instability
Demonstrable by formation of neoplastic lesions (adenoma and carcinoma)
Changes promote preneoplastic cells to
Although a number of nongenotoxic agents have been identified, no definitive mechanism(s) of action has been identified by which these nongenotoxic carcinogens function. One common effect of most nongenotoxic carcinogens is the induction of cell proliferation in the target tissue. The increase in cell proliferation has been shown to occur through either an increase in mitosis and DNA synthesis and/or a depression of apoptosis.
The cell proliferative response elicited by nongenotoxic carcinogens may result from interaction with cellular receptors, modulation of growth factors and disruption of cell growth regulation, regenerative growth secondary to necrosis, and/or inhibition of apoptosis (Bursch et al., 1984). Chemical compounds that induce cell proliferation can be divided into those that function through mitogenic mechanisms and those that induce cytolethality with a resultant compensatory hyperplasia (Butterworth, 1990). Chloroform is an example of the latter. Chloroform has been shown to induce mouse liver tumors only at doses of compound that produce liver necrosis, thus demonstrating an association between necrosis with compensatory hyperplasia and the resulting tumorigenicity. Mitogenic nongenotoxic compounds such as phenobarbital typically do not increase cell necrosis or cytolethality. While receptor-mediated changes have been reported with selective members of this group, for the most part, the mechanism of induction of DNA synthesis and cell proliferation by mitogenic nongenotoxic compounds has not been thoroughly defined.
replicative DNA synthesis and subsequent cell division has been linked to each of the stages of the
carcinogenesis process and has been suggested as the mechanism by which many nongenotoxic
carcinogens induce tumors (Pitot et al., 1981; Marsman et al., 1988; Buttterworth, 1990) (Figure 1).
increased DNA synthesis, two possible scenarios have been proposed for the induction of cancer.
In one, the less regulated increase in DNA synthesis and mitosis by the nongenotoxic agent may
induce mutations in dividing cells through misrepair. This coupled with continual cell division
pressure may serve to "fix" heritable mutations in a cell resulting in an initiated preneoplastic cell
that through additional cell proliferation pressure clonally expands into a preneoplastic lesion and
eventually a neoplasm. Alternately, the induction of DNA synthesis and mitosis by the
nongenotoxic agents may serve to allow the selective clonal growth of already "spontaneously initiated
cells" (Stott et al., 1988; Ames and Gold, 1990; Columbano et al., 1996). In this latter case,
the nongenotoxic agents would serve to promote the expansion of initiated or preneoplastic cells as
part of the tumor promotion phase of the multi-step carcinogenesis process (Farber, 1980) (Figure 1).
Tumor promotion has been associated with a sustained induction of cell replication (Slaga,
1983, 1984). The carcinogenicity of many compounds including phenobarbital, cyproterone acetate,
and ethinylestradiol (Schulte-Hermann et al., 1983; Schulte-Hermann, 1987) appear to act through a
cell proliferation mechanism that is dependent on the continued administration of the compound.
Under sustained administration of a tumor promoter, an increase in the number of preneoplastic foci
has been reported. Both of these scenarios, formation of new initiated cells and clonal expansion
of already present preneoplastic cells apparently require sustained and chronic induction of cell
proliferation and DNA synthesis for tumorigenesis to proceed.
Figure 1. Stages of Hepatic Carcinogenesis
Interestingly, while an increase in cell proliferation by nongenotoxic agents has been demonstrated with a number of epigenetic agents, many of these studies have shown a temporal, 1-2 week increase in cell proliferation, followed by a return to normal mitotic activity in the target tissue even though the compound is still being administered. Thus the apparent need for sustained and chronic DNA synthesis may not be fulfilled. This apparent paradox may be explained by several findings. One, while the apparent initial burst of DNA synthetic activity is temporal, closer examination has revealed that in many cases a slight increase in DNA synthetic activity over control levels is maintained or a sub-population of cells (midzonal or centrolobular cells in the liver) show a sustained increase (Bahneman, personnel communication). Secondly, work by our group and others have shown that for several liver nongenotoxic agents, a temporal increase in DNA synthesis occurs in the normal liver but a more sustained increase in DNA synthesis is found in preneoplastic cells, thus suggesting a tumor promotional effect of these agents (Kolaja et al 1996 a,b).
In concert with the selective stimulation of cell proliferation seen in preneoplastic cells by the nongenotoxic carcinogens, some nongenotoxic agents appear to have "mito-inhibitory" effects on normal cells (non-initiated cells). This mito-suppression in the liver is characterized by a reduction in basal/normal hepatocellular turnover and/or capacity to be stimulated to proliferate in vivo or in vitro (Yager et al., 1995; Eckl et al., 1988; Tsai et al., 1991). These findings support the resistant hepatocyte model as proposed by Farber (1990) in that preneoplastic focal lesions undergoing clonal expansion arise from initiated hepatocytes that are resistant to growth regulatory control. The mechanisms of mito-suppression by nongenotoxic carcinogens are not established. The mito-suppression observed following sustained treatment with phenobarbital may involve increased transforming growth factor b (TGF-b) and manose-6-phosphate/insulin-like growth factor II receptor (involved with uptake of TGB-b) expression (Jirtle et al., 1994). A variety of growth factors, however, appear to be involved in stimulating hepatocellular DNA synthesis. Complete mitogens, epidermal growth factor (EGF), transforming growth factor, hepatocyte growth factor, hepatic stimulatory substance, hepatoprotein B, and heparin binding growth factor-1 can directly stimulate DNA replication in the liver. Other growth factors such as norepinephrine, estrogens, insulin and glucagon, enhance the growth stimulatory effects of direct mitogens and have been termed comitogens (Michalopoulos, 1990). Growth inhibitors in the liver include transforming growth factor b (TGFb), interleukin 1b and hepatocyte proliferation inhibitor. The involvement of these growth factors and growth factor receptors in the cell proliferation associated with exposure to nongenotoxic hepatocarcinogens requires additional study. In addition, recent studies in the liver have been reported that suggest a role for the Kupffer cell in the modulation of cell proliferation through tumor necrosis factor a release. The Kupffer cell has also been shown to play a role in modulating and inducing oxidative stress injury in the liver. Further information is necessary on what role this cell type plays in the modulation of cell proliferation and hepatocellular neoplasia. While most investigators agree that the induction of cell proliferation is an integral component of chemically induced carcinogenesis and is fairly predictive of nongenotoxic carcinogenesis, others have questioned the premise that enhancement of cell division plays a major role in the cancer process by nongenotoxic agents (Melnick, et al., 1992; Melnick and Huff, 1993).
While the linkage between the induction of cell proliferation and the carcinogenesis
process is strong with epigenetic carcinogens, exact mechanisms for the increase in cell proliferation and
the selective clonal growth of preneoplastic initiated cells following exposure to nongenotoxic
agents remains unclear. Exposure to nongenotoxic carcinogens has other cellular effects. The role of
some of these cellular events including inhibition of apoptosis, induction of oxidative stress, inhibition
of gap junctional intercellular communication, and altered gene expression that are likely to
contribute to the carcinogenesis process by nongenotoxic chemicals are discussed below.
Apoptosis is a normal mechanistic process in which cells that acquire/receive unrepairable severe damage to DNA or other cellular macromolecules, or cells with a low apoptotic threshold are selectively removed from the tissue. In maintaining cell number within a tissue, a dynamic equilibrium exists between mitosis and apoptosis, such that these processes are balanced. With respect to carcinogenesis, it is generally accepted that the cancer process is a result of an imbalance between cell growth and death. Thus, the apoptotic process serves as a cellular mechanism to counter aberrant proliferation. The inhibition of apoptosis has been linked to tumor promotion in vivo. In an initiation-promotion rat liver model of carcinogenesis, chronic treatment with many nongenotoxic hepatocarcinogens including phenobarbital results in an increase rate of formation, and number of foci (Schulte-Hermann et al., 1990). This occurs predominantly through an inhibition of apoptosis in the liver foci (Schulte-Hermann, et al., 1994). This inhibition of apoptosis has also been shown in vitro by the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA), by ionizing radiation, and acute serum deprivation (Tomei et al., 1988). Several tumor promoters appear to act by invoking a resistance to apoptosis in initiated cells thus allowing for clonal expansion of altered cells.
Consistent with the concept that liver tumor promoters produce effects that are reversible upon removal of the compound, withdrawal of liver tumor promoters results in an enhanced rate of apoptosis and ultimately restores the liver to its original cell number (Columbano et al., 1996). Also, initiated cells appear to be preferentially deleted by apoptosis following removal of tumor promoters (Schulte-Hermann et al., 1995; Bursch et al., 1984, Kolaja et al, 1996a,b). The increase in apoptosis often seen concomitantly with increases in cell proliferation by liver tumor promoters has been proposed as protective mechanism that functions to delete genetically damaged cells prior to cell replication. This line of reasoning is further supported from results with dietary restriction. Using the initiation-promotion model of carcinogenesis, a negative growth effect on cell proliferation in the liver has been shown. Dietary restriction has been shown to inhibit hepatocellular cell proliferation and enhance apoptotic rates in focal lesions in mice and rats (Grasl-Kraupp et al., 1994; Kolaja et al., 1996c; Klaunig and Kamendulis, 1999). The effect of dietary restriction appears most effective at the tumor promotion stage of carcinogenesis. It has been hypothesized that the energy and or support factors in the dietary restricted animal may not be sufficient compared to non-dietary restricted animals in maintaining cell number. This results in increased apoptosis. Since initiated cells appear to be preferentially deleted by the apoptotic process, dietary restriction results in deletion of cells and may explain why dietary restriction inhibits tumor promotion. Collectively, these results are consistent with the concept that apoptosis may protect against carcinogenesis by deleting altered and potentially mutagenic cells and supports that inhibition of apoptosis is an important aspect of tumor promotion induced by chemical agents.
In addition, several oncogenes, tumor suppressor genes, and cell cycle regulatory genes
are linked to both the apoptotic process and propagation of altered cells leading to neoplasia.
Aberrant expression of the oncogene bcl-2, due in part to translocation of chromosomes 14 and 18, has
been shown to prevent apoptotic cell death (Cory, 1986). The tumor suppressor gene p53 regulates
the expression of genes that play a role in the G1/S phase of the cell cycle (Levine et al., 1994; Levine
et al., 1991). Loss or inactivation of gene function through mutation or deletion of the p53
tumor suppressor gene has been shown in more than 50% of human cancers (Hollstein et al., 1991)
and results in an inhibition of apoptosis. Furthermore, transfection and overexpression of wild-type
p53 into tumor cells inhibits cell proliferation and induces apoptosis (Shaw et al., 1992). The
apparent mechanism for regulation of apoptosis by p53 appears to involve a shift in the balance of Bcl-2
and Bax toward Bcl-2 such that the cell is primed to undergo apoptosis upon exposure to
apoptotic stimuli (Korsmeyer et al., 1993). Loss of p53 function during carcinogenesis may also
predispose preneoplastic cells to the accumulation of additional mutations by blocking normal apoptotic
responses to genetic damage. Thus, p53 deficiency may allow genetically altered cells to
escape deletion by apoptosis. Several oncogenic viruses promote the growth of virally-infected
cells through interfering with apoptotic genes. These include the adenovirus E1A and E1B oncogenes.
The E1A oncogene stimulates apoptosis in the absence of E1B, thus E1B inhibits
E1A-induced apoptosis (Rao et al., 1992). It has been proposed that E1B encodes for a viral analog of bcl-2
(Rao et al., 1992).
In multicellular organisms, the intercellular exchange of cellular factors from one cell to a neighboring cell is mediated through either extracellular signaling molecules (ie hormones) or between adjacent cells through gap junctional intercellular communication (Loewenstein, 1987; Pitts and Finbow, 1986). Gap junctions are comprised of plaques of transmembrane channels composed of connexin hexamers forming a connexin hemichannels (Revel and Karnovsky, 1967). The hemichannel of one cell connect with hemichannels on neighboring cells to form a transmembrane conduit between the two adjacent cells. This conduit allows for the exchange of ions and low molecular weight water soluble materials (less than 1000 daltons) between the cells. Growth regulatory signal transducing substances, including calcium, cAMP, and inositol triphosphate, substances that are involved in the regulation of the cell cycle, cell growth, and cell death are able to pass through the gap junction (Tsien and Weingart, 1976; Pitt and Sims, 1977; Cornell-Bell et al., 1990). Therefore, through gap junctions, a steady level of low molecular weight messenger molecules are maintained among cell populations.
Gap junctional intercellular communication has been shown to be modulated during the cancer process (Trosko and Ruch, 1998). Cell lines established from tumors of both animal and human tissues exhibit decreased gap junctional intercellular communication. In fact, during multistage rat hepatocarcinogenesis gap junctional intercellular communication has been shown to progressively decrease. Additionally, liver carcinomas removed from cancer patients have also displayed reduced ability to communicate with neighboring cells (Yamasaki, 1990; Krutovskikh and Yamasaki, 1997). Growth control in GJIC-deficient tumorogenic cells is restored by transfection with connexin genes (Mesnil and Yamasaki, 1993; Trosko and Chang, 1988).
Blockage of cell-to-cell communication has particularly been associated with the tumor promotion stage of chemical carcinogenesis and has been suggested as a mechanism of action for the tumor promotion process (Klaunig, 1991; Klaunig and Ruch, 1990; Williams, 1981; Budunova and Williams, 1984; Trosko and Chang, 1988; Klaunig 1991; Yamasaki, 1991). Many tumor promoting compounds demonstrate a tissue and species specific inhibition of gap junctional intercellular communication in vivo and in vitro following exposure (Klaunig and Ruch, 1987). A correlation between the ability of a compound to block cell-to-cell communication in cultured cells and its ability to induce rodent tumors through nongenotoxic mechanisms has also been demonstrated (Klaunig and Ruch, 1987; Trosko et al., 1982). Additionally, the inhibition of gap junctional intercellular communication observed following treatment with tumor promoting compounds correlates with species and strain sensitivity of the chemical (Diwan et al., 1985; Klaunig et al., 1990; Ruch and Klaunig 1988). Additional support for the involvement of gap junctional intercellular communication in the carcinogenesis process comes from studies examining anti-tumor promoting compounds and/or cancer chemopreventive agents. Several chemopreventive agents including the antioxidants vitamin E and the polyphenolic fraction of green tea, EGCG, have been reported to abolish the inhibition of GJIC observed following treatment with nongenotoxic/liver tumor promoting compounds (Trosko, 1997).
The mechanism for the involvement of gap junctional intercellular communication in
carcinogenesis process may relate to the fact that the gap junction mediates the passage of
both positive and negative growth regulatory molecules between neighboring cells. Therefore,
blockage of gap junctional intercellular communication between normal and preneoplastic cells creates
an environment in which preneoplastic cells are isolated from growth controlling factors of
normal surrounding cells (Klaunig and Ruch, 1990). Inhibition of gap junctional intercellular
communication would therefore be expected to be involved in decreased regulation of homeostatic
growth control allowing the preneoplastic cell to escape the growth control of normal surrounding
cells resulting in clonal expansion.
A number of xenobiotics produce liver tumors in rodents but have been considered of doubtful significance to man. The mechanism(s) for the species-selectivity, while realized, are unknown. One possible mechanism involved in the cancer process may be xenobiotic metabolism. Metabolism may result in the formation of reactive intermediates that can bind to DNA and result in base changes. Additionally, the compound or metabolite may induce DNA transcription or interaction with cellular receptors which may activate oncogenes, induce DNA replication, mitosis and cell proliferation.
In addition to these potential direct actions, a further series of indirect events associated with metabolism may be involved in carcinogenesis. Central to these actions is the production of reactive oxygen radicals. Metabolic activation and production of reactive oxygen species by cytochromes P450 is associated with many chronic diseases including chemical carcinogenesis and is related to DNA damage, activation of protein kinase C, and oncogenes, hyperplasia and in the metastatic process (Parke, 1994). Reactive oxygen species can be produced from many possible sources during metabolism; through redox cycling in the presence of molecular oxygen, through peroxidase-catalyzed single electron drug oxidations, and through futile cycling of cytochromes P450. Reactive oxygen species production (superoxide anion and hydrogen peroxide) may be greatly increased by futile cycling of the cytochromes P450. Of the potential sources of metabolism-derived reactive oxygen species, futile cycling of the cytochromes P450 greatly enhances reactive oxygen species (Parke and Ioannades, 1990). P450 futile cycling is especially seen with cytochrome P450 2E1. This enzyme is involved in the oxygenation of difficult to oxidize substrates including ethanol and thus generates reactive oxygen species in proximity of the substrate (Eksrom and Ingleman-Sundberg, 1989; Kuklielka and Cederbaum, 1992). Stabilization of 2E1 protein leads to a prolonged burst of reactive oxygen species production that may result in tissue necrosis, mutation and malignancy.
Induction of cytochrome P450 has been suggested as a predictive marker of potential
chemical carcinogenesis perhaps due to increased reactive oxygen species production. A common
feature of chemicals that induce mouse liver tumors or promote the growth of previously initiated rat
liver foci is that they induce cytochrome P450 2B (Rice et al., 1994; Lubet et al., 1989).
Phenobarbital, the liver tumor promoter, induces and is metabolically oxygenated by P4502B, but with
difficulty, such that the catalytic cycle is uncoupled and superoxide anion is released (Ingelman-Sundberg
and Hagbjork, 1982). Thus, the correlation between P4502B induction and tumor promotion
following phenobarbital exposure may be related to futile cycling and reactive oxygen species
production (Rice et al., 1994). Other chemicals such as peroxisome proliferators are potent inducers of
the cytochrome P4504 family. Following exposure to peroxisome proliferators, an increase in
peroxide and subsequent reactive oxygen species is also suggested (Lake et al., 1984; Reddy et al., 1982).
P450 oxidation of xenobiotics is higher in the mouse than in rat or man (Lornez et al., 1984).
The ability to metabolize the carcinogen 7,12-dimethylbenzanthracene is inversely correlated
with longevity and body weight among several species (Schwartz and Moore, 1979). This again
suggests a role for metabolism-dependent reactive oxygen species generation in chronic disease.
Irrespective of their genesis during metabolism, reactive oxygen species may contribute to the cancer process
via the conversion of superoxide anion and hydrogen peroxide to ïOH in the presence of iron
(Fenton and Haber-Weiss reaction) to yield DNA damage. The oxidative DNA damage may then result
in the production of single- or double-stranded DNA breaks, base modifications or rearrangements.
Alternately, reactive oxygen species may modulate gene expression and result in altered
growth regulation (Hsie, et al., 1986; Troll and Weisner,1985; Kensler and Taffe, 1986; Kensler and
Living in aerobic conditions, mammals face potential deleterious effects of endogenously
and exogenously generated reactive oxygen species on tissues and cells. It is estimated that two kg
of superoxide are generated in humans per year (Halliwell, 1994). To counter this insult, a
defense system consisting of antioxidants and oxidative repair enzymes is found in mammals (Hochstein
and Atallah, 1988). When the cellular balance between prooxidants and antioxidants is in favor of
the former the result is oxidative stress (Sies, 1985). A role for oxidative stress in the pathogenesis
of many age-related diseases has been implicated, including cancer (Guyton and Kensler, 1993;
Trush and Kensler, 1991; Vuillaume, 1987; Witz, 1991). Reactive oxygen species are enzymatically
or non-enzymatically generated in cells via endogenous (aerobic metabolism, P450 metabolism.
and phagocytosis) and exogenous sources (xenobiotics, anoxia, etc) (Figure 2). Normally, most
molecular oxygen taken into the body is converted to CO2 and water. However, approximately 5%
of molecular oxygen is converted to reactive oxygen species through normal metabolic
processes (Barber and Harris, 1994). Xenobiotics can enhance the reactive oxygen species load on the cell
by directly generating or indirectly inducing increased amounts of reactive oxygen species. Both
the endogenous production of reactive oxygen species and the ability to detoxify these radicals
are controlled by genetic and lifestyle factors, thus the genetic-environmental interaction is important
in defining the response of the cell to agents that induce reactive oxygen species.
Figure 2. Reactive Oxygen Species Production and Disruption of Cellular Homeostasis
In rodents definite species/strain differences in spontaneous tumor incidence and sensitivity to chemical carcinogenesis have been shown (Drinkwater and Bennett, 1991; Diwan, et al., 1991). While the genetic mechanisms behind these differences are not clear, an involvement for the induction of oxidative stress and resulting oxidative damage has been suggested. Reactive oxygen species generation, antioxidant activity, oxidative DNA repair capability and reactive oxygen modulation of gene expression have been shown to be genetically dependent (Burcham, 1999). This is readily demonstrable in mammals where the basal balance of pro-oxidant production and antioxidant detoxification of reactive oxygen species is normally buffered. This buffering system appears to function until an exogenous or endogenous increase in reactive oxygen species formation occurs, which determine the sensitivity of hosts to carcinogenic stimuli. Species with a higher basal level of oxidative stress might be of greater risk to further oxidative damage when exposed to oxidative insults from xenobiotics compared to those species with a lower low basal level of oxidative stress. This species specific relationship of basal oxidative stress is also seen in aging where induction of reactive oxygen species by mitochondria and P450 enzymes inversely correlate with the longevity of rodent species (Ku et al., 1993; Parke and Sapota, 1996). In this context, a relationship between basal levels of oxidative DNA damage with the sensitivity to skin tumor promotion effects of TPA was reported in several mouse strains (Wei et al., 1993).
Nongenotoxic carcinogens can directly generate or indirectly induce the generation of reactive oxygen species in cells (Rice-Evans and Burdon, 1993). Many epigenetic agents including peroxisome proliferators, chlorinated compounds, radiation, metal ions, barbiturate, and phorbol ester have shown oxidative stress induction in vitro and in vivo (Klaunig et al., 1997). While not completely resolved, it appears that there are several ways in which epigenetic carcinogens can generate reactive oxygen species. As noted above, compounds that are metabolized by and induce cytochrome P450 enzymes (Parke and Sapota, 1996) may produce reactive oxygen species through futile cycling.
A role for oxidative stress in the induction of hepatocellular cancer in rodents by the diverse group of hepatic carcinogens that also induce peroxisomes (peroxisome proliferators) has also been proposed. Additional H2O2 will leak out of newly formed peroxisomes producing an increase in the redox insult in the cell (Rao and Reddy, 1991; Tamura et al., 1990; Wade et al., 1992). This hypothesis has not been proven and recent studies have suggested that while an increase in oxidative DNA damage is seen with peroxisome proliferators, this increase does not correlate with the relative peroxisome proliferating activity of the compound. These latter findings do not preclude a role for oxidative stress in the cancer induction by peroxisome proliferators; it simply removes a possible role for the peroxisome in the process.
How the induction of oxidative stress by nongenotoxic agents results in carcinogenic activity remains unresolved. Oxidative stress induced by nongenotoxic chemicals may interfere with the physiological process of cells via oxidative damage and gene regulation. Oxidative damage can occur in DNA, lipid and protein. Oxidative DNA damage may be critical in carcinogenesis if it results in extensive enough changes to induce gene mutations. However, the induction of oxidative stress may also regulate gene expression either directly through activation of gene transcriptional pathways or indirectly through hypomethylation.
Both genotoxic and nongenotoxic carcinogens can produce an increase in reactive oxygen species which may result in oxidative DNA
damage (Klaunig et al., 1997). 8-hydroxy-2í-deoxyguanosine is a common oxidative DNA
adduct formed that has been shown to correlate with gene mutation and cellular transformation (Kamiya
et al., 1992; Shibutani et al., 1991). Although DNA damage with resulting mutation may be
produced with reactive oxygen species generators, the induction of oxidative stress by
epigenetic/nongenotoxic carcinogens may also result in altered gene expression without mutation.
Nongenotoxic carcinogens may elicit their effects in cells through signaling pathways: kinase; a cAMP-mediated cascade; a calcium-calmodulin activated pathway; and intracellular signal transducers such as nitric oxide (Kerr et al., 1992). These modifications may result in either increased cell proliferation or selective cell death (apoptosis or necrosis) (Timblin et al., 1997; Kass, 1997; Klaunig et al., 1997). Calcium is a signaling factor responsible for regulating a wide range of cellular processes including cell proliferation, differentiation, and apoptosis (Berridge, 1994; Whitfield, 1992). The release of calcium may result in the activation of kinases, such as protein kinase C (PKC) (Larsson and Cerutti, 1989; Brawn et al., 1995). The activation of PKC has been demonstrated following exposure to TCDD, chlorinated hydrocarbons, TPA and other nongenotoxic carcinogens (Zorn et al., 1995; Kass, 1989; Jirtle and Meyer 1991; Brockenbrough, 1991). Transcription factors are small molecule weight proteins that can bind with promoter regions of genes and in turn regulate their transcription (Vellanoweth et al., 1994). Two transcription factors, nuclear factor k-B (NFkB) and activator protein-1 (AP-1), have been studied for their regulation by nongenotoxic agents. (NFkB) is a heterodimer consisting of a 50kD and a 65kD subunit. The activation of the (NFkB) occurs in response to extracellular stimuli, physical stress, metal ions, asbestos, alcohol, and chemical treatment (Martin et al., 1999; Hart et al., 1999; Lu et al., 1999; Radler-Pohl et al., 1993; Gilmour et al., 1997). The activation of NFkB appears to be involved in the mediation of cell proliferation and apoptosis (Martin et al., 1999). Similarly, a shift in the redox potential of cells by reactive oxygen generating agents has been shown to up regulate these transcription factors (Hutter et al., 1997; Demple, 1997). Increases in reactive oxygen species can translocate NFkB to nuclei by the removal of IkB from NFkB heterodimer (Baeuerle and Baltimore, 1988; Schreck et al., 1991), but may reduce the DNA binding activity of NFkB to target genes (Sun and Oberley ,1996). Similar effects on the activation of the heat-shock transcription factor have been observed (Jacquier-Sarlin and Polla, 1996). The activation of AP-1 has been reported in cells following exposure to nongenotoxic chemicals, such as carbon tetrachloride, TCDD, phenobarbital, cadmium, alcohol, asbestos and TPA (Zawaski et al., 1993; Puga et al., 1992; Pinkus et al., 1993; Hart et al., 1999; Lu et al., 1999; Gilmour et al., 1997; Radler-Pohl et al., 1993). Interestingly, modulation of both prooxidants and antioxidants affect the activation of AP-1 (Muller et al., 1997).
Modification of the methylation status of the cell has also been seen with
nongenotoxic caracinogen exposure. Changes in DNA methylation modify gene expression. Post-DNA
synthetic methylation of the 5 position on cytosine (5-methylcytosine; 5mC) is the only naturally
occurring modification to DNA in higher eukaryotes. In vertebrate organisms, approximately 4% of
the cytosine residues in the genome are modified to 5mC (Ehrlich et al., 1982). It is generally
accepted that 5mC in DNA can affect cellular processes including development and differentiation;
however, it is strongly evidenced that the presence of 5mC is involved in cancer development (Laird
and Jaenisch 1997). In tumor cells, the methylation pattern of various genes is often altered such
that both hypermethylation and hypomethylation become apparent (Goodman and Counts, 1995).
Also, during carcinogenesis, widespread hypomethylation of the genome as well as hypermethylation
in areas that are normally unmethylated is seen (Baylin et al., 1991; Counts and Goodman
1995; Counts et al., 1996). The degree of methylation within a gene correlates inversely with its
expression (Costello et al., 1994; Boyes and Bird 1991). Hypermethylation of genes is associated
with decreased gene expression or gene silencing. Important to the cancer process, tumor
suppressor genes are known to be hypermethylated and subsequently inactivated. It has been suggested
that carcinogens may cause errors in methylation and lead to the formation of cells with increased
tumorigenic potential (Boehm and Drahovsky, 1983). Conversely, hypomethylation can lead to
enhanced gene expression and has been associated with increased mutation rates (Chen et al., 1998).
Oncogenes have been shown to become hypomethylated and their expression amplified (Belinsky
et al., 1998; Counts and Goodman, 1995). Exposure of rats to hepatocarcinogens or a
methyl-deficient diet (which itself is known to induce tumor formation) has been shown to decrease hepatic levels
of S-adenosyl-methionine (SAM); (Griffin and Karran, 1993). A decrease in this co-factor
would therefore promote hypomethylation and subsequent expression of oncogenes. In rats fed a
diet deficient in choline or methyl donor groups, hypomethylation of c-myc, c-fos and
c-H-ras protooncogenes was seen and was associated with hepatocarcinogenesis (Wainfen and
Poirier, 1992). Also consistent with the role of methylation on the carcinogenesis process, administration
of SAM has been shown to inhibit hepatocarcinogenesis. In addition to changes in gene
expression, methylation also regulates chromosome stability. Changes in DNA methylation patterns can
alter chromatin structure and result in deletions, inversions and loss of chromosomes (Pogribny et
al., 1995). Clearly, a role for methylation and changes in gene expression in the cancer process
is supported. Involvement of hypomethylation in tumor promotion is further supported by
studies examining methylation status in C3H, C57Bl/6 and B6C3F1 mice. These species show a
differential susceptibility to liver tumorigenesis in vivo in that the C57Bl/6 is relatively resistant
to hepatocarcinogenesis compared with the C3H and B6C3F1. In these mouse strains, the
oncogenes c-myc, c-fos and c-H-ras were shown to be less methylated in C3H and B6C3F1 than in
C57Bl/6 mice (Counts et al., 1996; Counts, et al., 1997). Thus, in these mouse strains, the likelihood
for oncogene expression (degree of methylation) correlated directly with cancer susceptibility.
These findings may in part explain the species specificity observed with respect to carcinogenicity.
Additionally, hepatocarcinogens may elicit their results through aberrant gene expression induced
by changes in methylation patterns.
The induction of cancer by nongenotoxic/epigenetic carcinogens appears to function through non DNA reactive mechanisms of action exhibiting both threshold and dose response characteristics. As noted above, the cellular effects of nongenotoxic carcinogens many times involve modification and changes to normal cellular processes. These changes seen with cell proliferation, induction of oxidative stress, modification of cell-to-cell communication and modulation of gene expression require that a threshold modification to these cellular processes be overwhelmed to invoke a toxic and carcinogenic response. The epigenetic cellular effects seen with nongenotoxic carcinogens are reversible. In addition, many of the cellular changes seen with nongenotoxic carcinogens involve intracellular targets that reflect inducible protective systems in the cell. For example, the exposure of cells and tissues to low levels of oxidative stress induce oxidative DNA repair enzymes and antioxidant enzyme systems to combat the increased oxidative insult. Induction of changes in gene expression by nongenotoxic carcinogens may serve to stimulate cellular detoxification systems to combat the nongenotoxic insult. This is exemplified by the production of mitoinhibitory effects seen on normal cells with some hepatic nongenotoxic compounds. In addition, the induction of P450 enzymes while possibly contributing to the toxic response seen with nongenotoxic carcinogens at high doses, at low doses may serve to detoxify the agents. The previously reported epigenetic effects of nongenotoxic carcinogens serve to securely place chemicals that participate through these mechanisms in a different category for human risk evaluation. Studies to date have for the most part shown that the epigenetic effects seen with nongenotoxic carcinogens are not applicable to humans since either mode of action, dose required to produce the effect, or target tissue response is different in humans than that seen in the rodent species. These experimental conclusions are confirmed by epidemiological data on occupational or therapeutically exposed humans to nongenotoxic rodent carcinogen
In summary, while much work remains in the understanding of the modes and mechanisms of action of nongenotoxic carcinogens and the epigenetic effects of these agents, it is apparent that this category of chemicals are functionally different than those compounds which directly interact, mutate, and modify genomic DNA.
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