Chapter 8. Chemical Carcinogenesis

• The term cancer describes a subset of neoplastic lesions.
• A neoplasm is defined as a heritably altered, relatively autonomous growth of tissue with abnormal regulation of gene expression.
• Metastases are secondary growths of cells from the primary neoplasm.
• A carcinogen is an agent whose administration to previously untreated animals leads to a statistically significant increased incidence of neoplasms of one or more histogenetic types as compared with the incidence in appropriate untreated animals.
• Initiation requires one or more rounds of cell division for the “fixation” of the DNA damage.
• Promotion results from the selective functional enhancement of the initiated cell and its progeny by the continuous exposure to the promoting agent.
• Progression is the transition from early progeny of initiated cells to the biologically malignant cell population of the neoplasm.

Cancer is a disease of cellular mutation, proliferation, and aberrant cell growth. It ranks as one of the leading causes of death in the world. Multiple causes of cancer have been either firmly established or suggested, including infectious agents, radiation, and chemicals. Estimates suggest that 70 to 90 percent of all human cancers have a linkage to environmental, dietary, and behavioral factors.

Definitions

Table 8–1 lists definitions of terms commonly used in discussing chemical carcinogenesis. For benign neoplasms, the tissue of origin is frequently followed by the suffix “oma”; for example, a benign fibrous neoplasm would be termed fibroma, and a benign glandular epithelium termed an adenoma. Malignant neoplasms from epithelial origin are called carcinomas, whereas those derived from mesenchymal origin are referred to as sarcoma. Thus, a malignant neoplasm of fibrous tissue would be a fibrosarcoma, whereas that derived from bone would be an osteosarcoma.

Carcinogens may be genotoxic, meaning that they interact physically with DNA to damage or change its structure. Other carcinogens may change how DNA expresses information without modifying or directly damaging its structure, or may create a situation in a cell or tissue that makes it more susceptible to DNA damage from other sources. Chemicals belonging to this latter category are referred to as nongenotoxic carcinogens. Common features of genotoxic and nongenotoxic carcinogens are shown in Table 8–2.

The carcinogenesis process involves a series of definable and reproducible stages. Operationally, these stages have been defined as initiation, promotion, and progression (Figure 8–1).

Initiation

The first stage of the cancer process involves initiation, a process that is defined as a stable, heritable change. This stage is a rapid, irreversible process that results in a carcinogen-induced mutational event. Chemical and physical agents that interact with cellular components at this stage are referred to as initiators or initiating agents. Initiating agents lead to genetic changes including mutations and deletions. Chemical carcinogens that covalently bind to DNA and form adducts that result in mutations are initiating agents. The initiating event becomes “fixed” when the DNA damage is not correctly or completely repaired prior to DNA synthesis and cell division.

Once initiated cells are formed, their fate has multiple potential outcomes: (1) the initiated cell can remain in a static nondividing state; (2) the initiated cell may possess mutations incompatible with viability or normal function and be deleted through apoptotic mechanisms; or (3) the cell may undergo cell division resulting in the proliferation of the initiated cell.

Promotion

The second stage of the carcinogenesis process involves the selective clonal expansion of initiated cells to produce a preneoplastic lesion. This is referred to as the promotion stage of the carcinogenesis process. Both exogenous and endogenous agents that operate at this stage are referred to as tumor promoters. Tumor promoters act through several mechanisms involving gene expression changes that result in sustained cell proliferation, through increases in cell proliferation and/or the inhibition of apoptosis. Promotion is reversible on removal of the promoting agent, and the focal cells may return to single initiated cell thresholds. In addition, these agents demonstrate a well-documented threshold for their effects. Below a certain dose or frequency of application, tumor promoters are unable to induce cell proliferation. Tumor promoters generally show organ-specific effects, for example, a tumor promoter of the liver such as phenobarbital will not function as a tumor promoter in the skin or other tissues.

Progression

Progression involves the conversion of benign preneoplastic lesions into neoplastic cancer. In this stage, due to increase in DNA synthesis and cell proliferation in the preneoplastic lesions, additional genotoxic events may occur resulting in further DNA damage including chromosomal aberrations and translocations. The progression stage is irreversible in that neoplasm formation, whether benign or malignant, occurs. With the formation of neoplasia, an autonomous growth and/or lack of growth control is achieved. Spontaneous progression can occur from spontaneous karyotypic changes that occur in mitotically active initiated cells during promotion. An accumulation of nonrandom chromosomal aberrations and karyotypic instability are hallmarks of progression.

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

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).

Mutagenesis

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.

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.

DNA Repair Mechanisms

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.

Excision Repair

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

Polyaromatic hydrocarbons such as benzo(a)pyrene are found at high levels in charcoal broiled foods, cigarette smoke, and in diesel exhaust.

Alkylating Agents

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.

Inorganic Carcinogens

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.

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.

Cytotoxicity

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.

Receptor Mediated

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.

Hormonal Mode of Action

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.

Retroviruses

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.

DNA Viruses

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.

Proto-oncogenes

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.

Tumor-Suppressor Genes

Retinoblastoma (Rb) Gene

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.

P53 Gene

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.

Chemoprevention

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.

Short-Term Tests for Mutagenicity

Short-term tests for mutagenicity were developed to identify potentially carcinogenic chemicals based on their ability to induce mutations in DNA either in vivo or in vitro. The majority of these tests measure the mutagenicity of chemicals as a surrogate for carcinogenicity. Although usually very predictive of indirect action and direct action (if a metabolic source is provided), these tests routinely fail to detect nongenotoxic carcinogens.

In Vitro Gene Mutation Assays

The most widely used short-term test is the Ames assay. Salmonella typhimurium strains deficient in DNA repair and unable to synthesize histidine are treated with several dose levels of the test compound, after which reversion to the histidine-positive phenotype is ascertained.

The mouse lymphoma assay is a mutagenicity assay used to determine whether a chemical is capable of inducing mutation in eukaryotic cells. The ability of the cell cultures to acquire resistance to trifluorothymidine (the result of forward mutation at the thymidine kinase locus) is quantified. Another mammalian cell mutation assay, the Chinese hamster ovary (CHO) test, is also commonly used to assess the potential mutagenicity of chemicals. This assay uses the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene as the endpoint.

In Vivo Gene Mutation Assays

The in vivo tests have advantages over the in vitro test systems in that they take into account the whole animal processes such as absorption, tissue distribution, metabolism, and excretion of chemicals and their metabolites. The commonly used in vivo models include transgenic rodent mutation assay systems based on the genes of the lac operon, Muta™Mouse, and Big Blue®.

To detect mutations, following exposure of mice to test chemicals, mutations are analyzed in high molecular weight DNA isolated from the tissue under investigation. The ratio of mutants to the total population will provide a mutation frequency for each chemical and each organ tested. In vivo genotoxicity test systems will fail to identify nongenotoxic/non-DNA reactive compounds.

Chromosomal Alterations

Chromosomal alterations are quite common in malignantneoplasms. Both in vivo and in vitro assays are available to assess chromosomal alterations. To assess induction of chromosomal alterations, cells are harvested in their first mitotic division after the initiation of chemical exposure. Cells are stained with Giemsa and scored for completeness of karyotype (21 ± 2 chromosomes). The classes of aberrations recorded include breaks and terminal deletions, rearrangements and translocations, as well as despiralized chromosomes, and cells containing 10 or more aberrations.

Sister chromatid exchanges (SCEs) are a measure of DNA damage events that are associated with mutation induction and cancer. SCEs are a reflection of an interchange of DNA between different chromatids at homologous loci within a replicating chromosome. Second-division metaphase cells are scored to determine the frequency of SCE/cell for each dose level. Disruption of the DNA replication process or damage to chromosomes by chemicals can alter the genetic material distributed to either of the two daughter nuclei. When this occurs, the genetic material that is not incorporated into a new nucleus may form its own “micronucleus,” which is clearly visible with a microscope. For this assay, animals are exposed to chemicals and the frequency of micronucleated cells is determined at some specified time after treatment.

DNA Damage

Primary DNA damage represents possible pre-mutational events that can be detected using mammalian cells in culture or using rodent tissue. Unscheduled DNA synthesis (UDS) is a commonly used assay that measures the ability of a chemical to induce DNA lesions by measuring the increase in DNA repair. Among the available techniques is the measurement of DNA strand breaks both in vivo and in vitro.

Transformation Assays

Various in vitro test systems have been developed to assess the carcinogenic potential of chemicals. The C3H/10T1/2 cell line has been widely used in the transformation assays. It was originally derived from fibroblasts taken from the prostate of a C3H mouse embryo. The cells are approximately tetraploid but the chromosome number in the cells varies widely. As such, these cells are chromosomally abnormal and have already passed through some of the stages that might be involved in the production of a cancerous cell. On plating these cells, they will stop growing when their density is sufficiently high (contact growth inhibition). However, the contact inhibition can fail, resulting in cell piling forming a transformed colony. Therefore, following exposure to xenobiotics, this assay assesses carcinogenic potential based on the percentage of colonies that are transformed.

The most frequently used endpoint for cell transformation is morphological transformation of mammalian cell fibroblasts in culture. Transformation assays using Syrian hamster embryo (SHE) cells are available for the assessment of the carcinogenic potential of chemicals. The SHE cell assay measures carcinogenic potential of xenobiotics by assessing transformed colonies based on morphological criteria.

Chronic Testing for Carcinogenicity

Chronic (2-Year) Bioassay

Two-year studies over the lifespan of rodents remain the primary method by which chemicals or physical agents are identified as having the potential to be hazardous to humans. In the chronic bioassay, two or three dose levels (up to the maximum tolerated dose, MTD) of a test chemical and a vehicle control are administered to 50 males and 50 females (mice and rats), beginning at 8 weeks of age, continuing throughout their lifespan. During the study, food consumption and bodyweight gain are monitored, and the animals are observed clinically on a regular basis, and at necropsy the tumor number, location, and pathological diagnosis for each animal is thoroughly assessed.

Organ-Specific Bioassays and Multistage Animal Models

Many tissue-specific bioassays have been developed with the underlying goal being to produce a sensitive and reliable assay that could be conducted in a time frame shorter in duration than the 2-year chronic bioassay. These assays are commonly used to detect carcinogenic activity of chemicals in various target organs.

Carcinogenicity Testing in the Liver

The liver represents a major target organ for chemical carcinogens. It has been estimated that nearly half of the chemicals tested in the 2-year chronic bioassay by the National Toxicology Program showed an increased incidence of liver cancer. Liver carcinogenesis assays have been developed to study and distinguish chemicals that affect the initiation or promotion stage of hepatocarcinogenesis. The ability of the test chemical to promote the growth of preneoplastic lesions can be assessed.

Carcinogenicity Testing in the Skin

The mouse skin model has been used to dissect mechanisms of carcinogenesis and also is purported to be a useful intermediate-term cancer bioassay. This model exploits many of the unique properties of the mouse skin, one major advantage being that the development of neoplasia can be followed visually. In addition, the number and relative size of papillomas and carcinomas can be quantified as the tumors progress. Both initiating and promoting activities of chemical carcinogens can be assessed using this model. Grossly, initiated cells of the skin appear identical to normal skin. Because the terminally differentiated cells in the skin are no longer capable of undergoing cell division, only initiated cells retain their proliferative capacity and thus represent the cell populations that give rise to tumors. On repeated application of tumor promoters, selective clonal expansion of initiated keratinocytes occurs, resulting in skin papillomas, which over time can progress to carcinomas.

Carcinogenicity Testing in Other Organs

Test systems to examine the ability of a chemical to promote neoplastic development at organ sites other than liver and skin have also been developed. The available systems include animal models directed at examining carcinogenicity in the lung, kidney, bladder, pancreas, stomach, colon, small intestine, and oral cavity. These models vary in the initiating carcinogen used, and frequency, duration, and site of application, as well as the duration of promoting chemical exposure.

Transgenic Animals in Carcinogenicity Assessment

Animal models with genetic alterations that invoke a susceptibility to carcinogenesis by chemical agents include Tg.AC and rasH2 transgenic mice, and p53+/– and XPA–/– knockout mice. Recently, the feasibility of the use of these animal models as alternative assays for the 2-year chronic bioassay was assessed by the Health and Environmental Sciences Institute (HESI) branch of the International Life Sciences Institute (ILSI). The conclusions drawn from the scientific review suggested that these models appear to have usefulness as screening models for assessment of chemical carcinogenicity; however, they do not provide definitive proof of potential human carcinogenicity. Further, the scientific panel suggested that these models could be used in place of the mouse 2-year bioassay. Coupled with information on genotoxicity, particularly DNA reactivity, structure–activity relationships, results from other bioassays, and the results of other mechanistic investigations including toxicokinetics, metabolism, and mechanistic information, these alternate mouse models for carcinogenicity appear to be useful models for assessing the carcinogenicity of chemical agents.

Many factors have been implicated in the induction of cancer in humans. Infectious agents, lifestyle, medical treatments, and environmental and occupational exposure account either directly or indirectly for the majority of cancers seen in humans. Of these, the component that contributes the most to human cancer induction and progression is lifestyle: tobacco use, alcohol use, and poor diet (Table 8–7). Tobacco usage either through smoking tobacco, chewing tobacco, or tobacco snuff-type products is estimated to be responsible for 25 to 40 percent of all human cancers. In particular, a strong correlation between tobacco usage and mouth, larynx, lung, esophageal, and bladder cancer exists. It has been estimated that 85 to 90 percent of all lung cancer cases in the United States are a direct result of tobacco use. The induction of pancreatic cancer also appears to have a linkage to tobacco use. Alcohol consumption contributes anywhere from 2 to 4 percent of cancers of the esophagus, liver, and larynx.

Poor diets, occupational exposures, and chemotherapeutic therapy account for many human cancers. High-fat and high-calorie diets have been linked to breast, colon, and gallbladder cancer in humans. Diets poor in antioxidants and/or vitamins such as vitamin A and vitamin E probably also contribute to the onset of cancer. The method of cooking may also influence the production of carcinogens produced in the cooking process. Carcinogenic heterocyclic amines and polycyclic aromatic hydrocarbons are formed during broiling and grilling of meat. Acrylamide, a suspected human carcinogen, has been found in fried foods at low concentrations. A number of occupations associated with the development of specific cancers are listed in Table 8–8. A number of medical therapeutic and diagnostic tools have also been linked to the induction of human cancer (Table 8–9). Therapeutic immunosuppression given to transplant patients or arising secondary to selective diseases such as acquired immune deficiency syndrome (AIDS) result in an increase in a variety of different neoplasms. These results further support the role of the immune system in identifying and removing early preneoplastic cells from the body.

Classification Evaluation of Carcinogenicity in Humans

The assessment and designation of a chemical or a mixture of chemicals as carcinogenic in humans is evaluated by various agencies worldwide. The evaluation usually encompasses both epidemiological and experimental animal and in vitro data utilizing assays as described earlier in this chapter. One of the first devised schemes for the classification of an agent's carcinogenicity was devised by the International Agency for Research on Cancer (IARC) (Table 8–10). The IARC approach assigns the chemical or mixture to one of five groupings based on strength of evidence for the agent's possible, probable, or definite carcinogenicity to humans. Similar classifications exist for the U.S. EPA, the Food & Drug Administration, and the European Community (EC). The classification of agents with regard to human carcinogenicity can be very difficult, in particular when animal data and/or epidemiological data in humans are inconclusive or confounded.