The development of neoplasia requires two major events: the formation of an initiated, mutated cell and the selective proliferation of the mutated cell to form a neoplasm. Chemicals that induce cancer have been broadly classified into one of two categories—genotoxic or DNA reactive and nongenotoxic or epigenetic carcinogens—based on their relative abilities to interact with genomic DNA.
Genotoxic carcinogens initiate tumors by producing DNA damage. DNA reactive carcinogens can be further subdivided according to whether they are active in their parent form (i.e., direct-acting carcinogens—agents that can directly bind to DNA without being metabolized) and those that require metabolic activation (i.e., indirect-acting carcinogens—compounds that require metabolism in order to react with DNA).
Direct-Acting (Activation-Independent) Carcinogens
Direct-acting carcinogens are highly reactive electrophilic molecules that can interact with and bind to nucleophiles, such as cellular macromolecules including DNA. Generally, these highly reactive chemicals frequently result in tumor formation at the site of chemical exposure.
The relative carcinogenic strength of direct-acting carcinogens depends in part on the relative rates of interaction between the chemical and genomic DNA, as well as competing reactions with the chemical and other cellular nucleophiles. Chemical stability, transport, and membrane permeability determine the carcinogenic activity of the chemical. Direct-acting carcinogens are typically carcinogenic at multiple sites and in all species examined.
Indirect-Acting Genotoxic Carcinogens
The majority of DNA reactive carcinogens are found as parent compounds, or procarcinogens, chemicals that require subsequent metabolism to be carcinogenic. Terms have been coined to define the parent compound (procarcinogen) and its metabolite form, either intermediate (proximate carcinogen) or final (ultimate carcinogen), that reacts with DNA. The ultimate form of the carcinogen is most likely the chemical species that results in mutation and neoplastic transformation. It is important to note that besides activation of the procarcinogen to a DNA reactive form, detoxification pathways may also occur resulting in inactivation of the carcinogen.
Indirect-acting genotoxic carcinogens usually produce their neoplastic effects at the target tissue where the metabolic activation of the chemical occurs and not at the site of exposure (as seen with direct-acting genotoxic carcinogens).
Effects of mutations depend on when in the cell cycle the adducts are formed, where the adducts are formed, and the type of repair process used in response to the damage. Mutagenesis may result from misread DNA, frame-shifting, or broken DNA strands.
Damage by Alkylating Electrophiles
Electrophiles can readily form covalent adducts with nucleophilic targets. Because of their unpaired electrons, S, O, and N atoms are nucleophilic targets of many electrophiles. In general, the stronger electrophiles display a greater range of nucleophilic targets, whereas weak electrophiles are only capable of alkylating strong nucleophiles.
An important and abundant source of nucleophiles is contained not only in the DNA bases, but also in the phosphodiester backbone. Different electrophilic carcinogens will often display different preferences for nucleophilic sites in DNA and different spectra of damage.
Another common modification to DNA is the hydroxylation of DNA bases. Oxidative DNA adducts have been identified in all four DNA bases. The source of oxidative DNA damage is typically formed from free radical reactions that occur endogenously in the cell or from exogenous sources.
Methylation of DNA results in heritable expression or repression of genes, with hypomethylation associated with active transcription of genes, whereas hypermethylated genes tend to be rarely transcribed. Chemical carcinogens may inhibit DNA methylation by forming covalent adducts, single-strand breaks in the DNA, alteration of methionine pools, and inactivation of the DNA methyltransferase responsible for methylation. Whether a particular DNA adduct will result in mutation depends in part on the process of DNA replication and in part on DNA repair.
Following the formation of a carcinogen DNA adduct, the persistence of the adduct is a major determinant of the outcome. This persistence depends on the ability of the cell to repair the altered DNA. However, the presence of a DNA adduct is not sufficient for the carcinogenesis process to proceed. The relative rates or persistence of particular DNA adducts may be an important determinant of carcinogenicity. Differences in susceptibility to carcinogenesis are likely the result of a number of factors, including DNA replication within a tissue and repair of a DNA adduct. The development of cancer following exposure to chemical carcinogens is a relatively rare event because of a cell's ability to recognize and repair damaged DNA. To be effective in restoring a cell to normal, repair of DNA must occur prior to cell division.
Although cells possess mechanisms to repair many types of DNA damage, these are not always completely effective, and residual DNA damage can lead to the synthesis of altered protein. Mutations in an oncogene, tumor-suppressor gene, or gene that controls the cell cycle can result in a clonal cell population with a survival advantage.
Cells have several mechanisms for repairing DNA damage. Repair of DNA damage does not always occur prior to cell replication, and repair of DNA damage by some chemicals is relatively inefficient.
Mismatch Repair of Single-Base Mispairs
Many spontaneous mutations are point mutations, a change in a single-base pair in the DNA sequence. Depurination is a fairly common occurrence and a spontaneous event in mammals, and results in the formation of apurinic sites. All mammalian cells possess apurinic endonucleases that function to cut DNA near apurinic sites. The cut is then extended by exonucleases, and the resulting gap repaired by DNA polymerases and ligases.
DNA regions containing chemically modified bases, or DNA chemical adducts, are typically repaired by excision repair processes. Proteins that slide along the surface of a double-stranded DNA molecule recognize irregularities in the shape of the double helix, and induce repair of the lesion.
End-Joining Repair of Nonhomologous DNA
A cell that has double-strand breaks can be repaired by joining the free DNA ends. The joining of broken ends from different chromosomes, however, will lead to the translocation of DNA pieces from one chromosome to another, translocations that have the potential to enable abnormal cell growth. Homologous recombination is one of two mechanisms responsible for the repair of double-strand breaks. In this process, the double-strand break on one chromosome is repaired using the information on the homologous, intact chromosome.
The predominant mechanism for double-stranded DNA repair in multicellular organisms is nonhomologous repair, which involves the rejoining of the ends of the two DNA molecules. Although this process yields a continuous double-stranded molecule, several base pairs are lost at the joining point. This type of deletion may produce a potentially mutagenic coding change.
Classes of Genotoxic Carcinogens
Polyaromatic hydrocarbons such as benzo(a)pyrene are found at high levels in charcoal broiled foods, cigarette smoke, and in diesel exhaust.
Alkylating chemicals represent an important class of chemical carcinogens. Whereas some alkylating chemicals are direct-acting genotoxic agents, many require metabolic activation to produce electrophilic metabolites that can react with DNA. Alkylating agents can be classified into several groups including direct-acting alkylalkanesulfonates (methyl- and ethyl methanesulfonate) and nitrosamides (N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea, N-methyl-N-nitro-N-nitrosoguanidine), and the indirect-acting nitrosamides (dimethyl- and diethylnitrosamines). The alkylating compounds readily react with DNA at more than 12 sites. The N7 position of guanine and the N3 position of adenine are the most reactive sites in DNA for alkylating chemicals.
Aromatic Amines and Amides
Aromatic amines and amides encompass a class of chemicals with varied structures. The aromatic amines undergo both phase-I (hydrolysis, reduction, and oxidation) and phase-II (conjugation) metabolism. Phase-I reactions occur mainly by cytochrome P450-mediated reactions, yielding hydroxylated metabolites that are often associated with adduct formation in proteins and DNA, and produce liver and bladder carcinogenicity.
Several metals exhibit carcinogenicity in experimental animals and/or exposed humans. Table 8–3 provides a listing of some common metals and their corresponding carcinogenicity in animals and humans. Additional discussion of selected metals is in Chapter 23.
Table 8–3 Carcinogenicity of metals. ||Download (.pdf)
Table 8–3 Carcinogenicity of metals.
Mice, dogs, rats
Drinking water (oral)
Mice, rats, monkeys
Mice, rats, chickens
Mice, rats, rabbits
Hamsters, mice, rats, rabbits
Mice, rats, cats, hamsters, rabbits
Gastric and renal carcinoma
Chickens, rats, hamsters
Nongenotoxic (Epigenetic) Carcinogens
A number of chemicals that produce tumors in experimental animals following chronic treatment appear to act via mechanisms not involving direct binding, damage, or interaction of the chemical or its metabolites with DNA. These agents have been labeled nongenotoxic carcinogens. The targets induced by nongenotoxic carcinogens are often in tissues where a significant incidence of background, spontaneous tumors is seen in the animal model. Prolonged exposure to relatively high levels of chemicals is usually necessary for the production of tumors. The diverse biochemical modes of action for non-DNA reactive carcinogens are noted in Table 8–4.
Table 8–4 Proposed modes of action for selected nongenotoxic chemical carcinogens. ||Download (.pdf)
Table 8–4 Proposed modes of action for selected nongenotoxic chemical carcinogens.
Mode of Action
Fibrates (e.g., clofibrate)
Polychlorinated biphenyls (PCBs)
Polybrominated biphenyls (PBBs)
Steroid and peptide hormones
Oxidative stress inducers
Chemicals that function through this mechanism produce sustained cell death. Often, metabolism of the chemical is accompanied by persistent regenerative growth, resulting in the potential for the acquisition of “spontaneous” DNA mutations and allowing mutated cells to accumulate and proliferate. This process then gives rise to preneoplastic focal lesions that on expansion can lead to tumor formation. The induction of cytotoxicity may be observed with many carcinogens both genotoxic and nongenotoxic when high toxic exposures occur. Thus, the induction of cytotoxicity with compensatory hyperplasia may contribute to the observed tumorigenicity of many carcinogenic chemicals at high doses.
P450 Inducers: Phenobarbital-Like Carcinogens
Phenobarbital is a commonly studied non-DNA reactive compound that is known to cause tumors by a nongenotoxic mechanism involving liver hyperplasia. The induction of CYP2B by phenobarbital is mediated by activation of the constitutive androstane receptor (CAR), a member of the nuclear receptor family. Other CAR-dependent phenobarbital responses that are critical for tumor formation include increased cell proliferation, inhibition of apoptosis, inhibition of gap junctional communication, hypertrophy, and development of preneoplastic focal lesions in the liver.
Peroxisome Proliferator-Activated Receptor α (PPARα)
Various chemicals are capable of increasing the number and volume of peroxisomes in the cytoplasm of cells. These peroxisome proliferators include chemicals such as herbicides, chlorinated solvents (e.g., trichloroethylene and perchloroethylene), plasticizers (e.g., diethylhexylphthalate and other phthalates), lipid-lowering fibrate drugs (e.g., ciprofibrate and clofibrate), and natural products. In addition, many of these chemicals produce liver enlargement and hepatocellular carcinoma in rats and mice through non-DNA reactive mechanisms. The currently accepted mode of action for this class of chemicals involves agonist binding to the nuclear hormone receptor, PPARα. PPARα is highly expressed in cells that have active fatty acid oxidation capacity. PPARα plays a central role in lipid metabolism and acts as a transcription factor to modulate gene expression following ligand activation.
Hormonally active chemicals include biogenic amines, steroids, and peptide hormones that cause tissue-specific changes through interaction with a receptor. Trophic hormones are known to induce cell proliferation at their target organs. This action may lead to the development of tumors when the mechanisms of hormonal control are disrupted and some hormone shows persistently increased levels.
Estrogenic agents can induce tumors in estrogen-dependent tissue. Individuals with higher circulating estrogen levels and those with exposure to the potent estrogenic agent diethylstilbesterol (DES) are at increased risk of cancer development. DES has been causally linked to the higher incidence of adenocarcinomas of the vagina and cervix in daughters of women treated with the hormone during pregnancy. The effects of steroidal chemicals on the cell cycle and on microtubule assembly may be important in the aneuploidy inducing effects of some hormonal agents.
A number of chemicals that reduce thyroid hormone concentrations (T4 and/or T3) and increase thyroid-stimulating hormone (TSH) have been shown to induce neoplasia in the rodent thyroid. TSH demonstrates proliferative activity in the thyroid, with chronic drug-induced TSH increases leading to progression of follicular cell hypertrophy, hyperplasia, and eventually neoplasia.
DNA Methylation and Carcinogenesis
The degree of methylation within a gene inversely correlates with the expression of that gene. Several chemical carcinogens are known to modify DNA methylation, methyltransferase activity, and chromosomal structure. During carcinogenesis, both hypomethylation and hypermethylation of the genome have been observed. Tumor-suppressor genes have been reported to be hypermethylated in tumors. Hypomethylation has been associated with increased mutation rates because many oncogenes are hypomethylated and their expression is amplified.
Reactive oxygen species have also been shown to modify DNA methylation by interfering with the ability of methyltransferases to interact with DNA; the resulting hypomethylation allow the expression of normally quiescent genes. Also, the abnormal methylation pattern observed in cells transformed by chemical oxidants may contribute to an overall aberrant gene expression and promote tumorigenesis.
Oxidative Stress and Chemical Carcinogenesis
Oxygen radicals can be produced by both endogenous and exogenous sources and are typically counterbalanced by antioxidants. Antioxidant defenses are both enzymatic (e.g., superoxide dismutase, glutathione peroxidase, and catalase) and nonenzymatic (e.g., vitamin E, vitamin C, β-carotene, and glutathione). Endogenous sources of reactive oxygen species include oxidative phosphorylation, P450 metabolism, peroxisomes, and inflammatory cell activation. Through these or other currently unknown mechanisms, a number of chemicals that induce cancer (e.g., chlorinated compounds, radiation, metal ions, barbiturates, and some PPARα agonists) induce reactive oxygen species formation and/or oxidative stress.
Oxidative Damage and Carcinogenesis
Reactive oxygen species left unbalanced by antioxidants can result in damage to cellular macromolecules. In DNA, reactive oxygen species can produce single- or double-stranded DNA breaks, purine, pyrimidine, or deoxyribose modifications, and DNA crosslinks.
Mutations and oxidative damage to mitochondrial DNA have been identified in a number of cancers. Compared with nuclear DNA, mitochondrial genome is relatively susceptible to oxidative base damage due to (1) close proximity to the electron transport system, a major source of reactive oxygen species; (2) mitochondrial DNA is not protected by histones; and (3) DNA repair capacity is limited in the mitochondria.
Oxidative Stress and Cell Growth Regulation
Activation of signaling cascades by reactive oxygen species induced by chemical carcinogens ultimately leads to altered gene expression for a number of genes including those affecting proliferation, differentiation, and apoptosis. Activation of NFκB, a ubiquitously expressed transcription factor, is regulated, in part, by reactive oxygen species and the cellular redox status, and has been observed to occur following a wide variety of extracellular stimuli, including exposure to chemical carcinogens such as PPARα agonists and PCBs.
Gap Junctional Intercellular Communication and Carcinogenesis
Gap junctional intercellular communication appears to play an important role in the regulation of cell growth and cell death, in part through the ability to exchange small molecules (<1 kDa) between cells. If cell communication is blocked between tumor and normal cells, the exchange of growth inhibitory signals from normal cells to initiated cells is prevented thus allowing the potential for unregulated growth and clonal expansion of initiated cell populations.
Polymorphisms in Carcinogen Metabolism and DNA Repair
Genetic polymorphisms arise from human genetic variability. In carcinogenesis, genetic polymorphisms may account for the susceptibility of some individuals to certain cancers. A number of polymorphisms have been described in carcinogen-metabolizing enzymes, with certain alleles linked to altered risk of selective cancers. Glutathione S-transferases (GSTs) are highly polymorphic in humans. The GSTM1 isoform is particularly important in carcinogenesis, because of its high reactivity toward epoxides.
Carcinogenic risk depends on both exposure (dose and duration) as well as genetic susceptibility. For example, if the genetic susceptibility is high, then exposure to a chemical carcinogen will result in a higher risk for cancer development.
Proto-oncogenes and Tumor-Suppressor Genes
Proto-oncogenes and tumor-suppressor genes encode a wide array of proteins that function to control cell growth and proliferation. Common characteristics of oncogenes and tumor-suppressor genes are shown in Table 8–5. Mutations in both oncogenes and tumor-suppressor genes contribute to the progressive development of human cancers. Accumulated damage to multiple oncogenes and/or tumor-suppressor genes can result in altered cell proliferation, differentiation, and/or survival of cancer cells.
Table 8–5 Characteristics of proto-oncogenes, cellular oncogenes, and tumor-suppressor genes. ||Download (.pdf)
Table 8–5 Characteristics of proto-oncogenes, cellular oncogenes, and tumor-suppressor genes.
Broad tissue specificity for cancer development
Broad tissue specificity for cancer development
Considerable tissue specificity for cancer development
Germ line inheritance rarely involved in cancer development
Germ line inheritance frequently involved in cancer development
Germ line inheritance frequently involved in cancer development
Analogous to certain viral oncogenes
No known analogs in oncogenic viruses
No known analogs in oncogenic viruses
Somatic mutations activated during all stages of carcinogenesis
Somatic mutations activated during all stages of carcinogenesis
Germ line mutations may initiate, but mutation to neoplasia occurs only during progression stage
The Rous sarcoma virus (RSV) is capable of transforming a normal cell and producing sarcomas. The genome of RSV and other retroviruses consists of two identical copies of mRNA, which is then reverse transcribed into DNA and incorporated into the host-cell genome. Oncogenic transforming viruses like RSV contain the v-src gene, a gene required for cancer induction. Normal cells contain a gene closely related to v-src in RSV. This discovery showed that cancer may be induced by the action of normal, or nearly normal, genes.
Infection by small DNA viruses is lethal to most nonhost animal cells; however, a small proportion integrates the viral DNA into the host-cell genome. The cells that survive infection become permanently transformed due to the presence of one or more oncogenes in the viral DNA. Papilloma viruses can infect and cause tumors in humans. Of the human papilloma viruses, types 16, 18, 31, and 33 are associated with human cervical cancers.
An oncogene encodes a protein that is capable of transforming cells in culture or inducing cancer in animals. Of the known oncogenes, the majority appear to have been derived from normal genes (i.e., proto-oncogenes), and are involved in cell signaling cascades. Because most proto-oncogenes are essential for maintaining viability, they are highly conserved. Activation of proto-oncogenes to oncogenes arises through mutational events occurring within proto-oncogenes. It has been recognized that a number of chemical carcinogens are capable of inducing mutations in proto-oncogenes. Oncogene products can operate at multiple levels of signaling cascades, including ligand, receptor, and transcription factor stages of transduction.
The proteins encoded by most tumor-suppressor genes act as inhibitors of cell proliferation or cell survival (Table 8–6). The prototype tumor-suppressor gene, Rb, was identified by studies of inheritance of retinoblastoma. Loss or mutational inactivation of Rb contributes to the development of a wide variety of human cancers. In its unphosphorylated form, Rb binds to the E2F transcription factors preventing E2F-mediated transcriptional activation of a number of genes whose products are required for DNA synthesis. Rb becomes phosphorylated during the late G1, causing dissociation from E2F—a process that allows E2F to induce synthesis of DNA replication enzymes, resulting in a commitment to the cell cycle.
Table 8–6 Examples of tumor-suppressor genes and cancer association. ||Download (.pdf)
Table 8–6 Examples of tumor-suppressor genes and cancer association.
Small-cell lung carcinoma
Breast, colon, lung cancers
The p53 protein is a tumor-suppressor gene that is essential for checkpoint control and arrests the cell cycle in G1 in cells with damaged DNA. Cells with functional p53 arrest in G1 when exposed to DNA damaging agents, whereas cells lacking functional p53 are unable to block the cell cycle. p53 is activated by a wide array of stressors including ultraviolet light, γ irradiation, heat, and several carcinogens.
In most cells, accumulation of p53 also leads to induction of proteins that promote apoptosis, and therefore would prevent proliferation of cells that are likely to accumulate multiple mutations. When the p53 checkpoint control does not operate properly, damaged DNA can replicate, producing mutations and DNA rearrangements that contribute to the development of transformed cells.
Hormesis and Carcinogenesis
Hormesis is defined as a dose–response curve in which a U-, J-, or inverted U-shaped dose–response is observed; with low-dose exposures often resulting in beneficial rather than harmful effects. Adaptive responses have been proposed to explain the hormetic effects observed by chemical carcinogens. These usually involve actions of the chemical on cellular signaling pathways that lead to changes in gene expression, resulting in enhanced detoxification and excretion of the chemical, as well as preserving the cell cycle and programmed cell death. It has been proposed that following very low doses of chemicals, the upregulation of these mechanisms overcompensates for cell injury such that a reduction in tumor promotion and/or tumor development is seen, and would explain the U- or J-shaped response curves obtained following carcinogen exposure. A common feature of chemical carcinogens for which hormetic effects have been proposed is the formation of reactive oxygen species and the induction of cytochrome P450 isoenzymes.
The study of chemicals that prevent, inhibit, or slow down the process of cancer is referred to as chemoprevention. A number of chemicals, including drugs, antioxidants, foodstuffs, and vitamins, have been found to inhibit or retard the components of the cancer process in both in vitro and in vivo models. A basic assumption in chemoprevention is that treating early stages of malignant process will halt or delay the progression to neoplasia. Chemopreventive agents may function as inhibitors of carcinogen formation, blocking agents, and/or suppressing agents. Blocking agents serve to prevent the metabolic activation of genotoxic or nongenotoxic carcinogens by either inhibiting its metabolism or by enhancing the detoxification mechanisms. Suppressing agents induce tissue differentiation, may counteract oncogenes, enhance tumor-suppressor gene activities, inhibit proliferation of premalignant cells, or modify the effect of the carcinogen on the target tissue.