15 James E. Klaunig In the United States, cancer ranks as the second leading cause of death. Over 1 million new cases of cancer are diagnosed each year and more than 1½ million Americans die yearly from cancer. Most human cancers surface in the fifth, sixth, and seventh decade of life. However, cancer, usually genetically related, is also seen in children and younger adults. Over 50% of American men and approximately one-third of American women will develop cancer during their lifetimes. The risk of developing most types of cancer can be reduced or at least delayed by lifestyle changes including limiting exposure to the sun, cessation of smoking, eating a healthy diet, and becoming more active physically. Besides lifestyle, other causes of cancer have been established including exposure to infectious agents, radiation, and xenobiotic chemicals. Estimates suggest that 70–90% of all human cancers have a linkage to environmental, dietary, and behavioral factors (Table 15.1). Cancer is a disease of cellular mutation and aberrant cell growth. Exposure to a chemical or physical agent is usually at the root of the cancer induction whether the cause of cancer is the result of lifestyle, occupational, pharmaceuticals, or environmental factors. While our understanding of the mechanisms by which a normal cell changes to a malignant cell has progressed considerably in the last four decades, we are still naïve to many of the steps involved in the cancer process and to the means to prevent and cure this disease. Table 15.1 Human Cancer Death Rates Linked to Various Exposure Factors This chapter will address: Historically, there has been an association of epidemiology between the induction of cancer in human and chemical exposure (Table 15.2). For example, Percival Pott noted in the latter part of the eighteenth century a cause-and-effect linkage between scrotal cancer and an occupation—chimney sweeps. Pott suggested that the daily exposure to chimney soot was directly related to the cancer induction in these workers. A 100 years later, Butlin following up on Potts work found that European chimney sweeps had a much lower incidence of scrotal cancer. He hypothesized that the difference was due to age of the workers at the start of the exposure (young boys in England, adults in Europe) as well as better hygienic habits of the European sweeps. These findings illustrate two important foundations of chemical carcinogenesis that relate to cancer induction and incidence. One factor is the relationship between age of exposure and cancer induction, whereby young subjects are more sensitive to the carcinogenic effects of a chemical. A second factor is the duration of exposure, where exposure to the British sweeps began in their youth and thus lasted longer in duration than the European sweeps initially exposed as adults. Rehn, in another occupational study in the late 1800s showed a linkage between the manufacturer of aniline dyes used for dyeing fabric and the induction of bladder cancer in dye workers. Subsequent work in the twentieth century showed that aniline dyes contained aromatic amine compounds (2-napthylamine and benzidine) that were responsible for the association between bladder cancer in humans and exposure to aniline dyes and helped to define the specific chemicals in the dyes responsible for the cancer induction. These chemicals were later found to react with nuclear DNA and to produce mutations in the DNA following metabolite modification. The observed epidemiological studies on cancer induction in humans following exposure to chemicals and radiation gave rise to laboratory studies, frequently using rodents as the model to further understand the cause and pathological changes seen in the cancer process. The development of these rodent models was paramount in the development of the multistage cancer models used today and to the discovery of therapeutic and prevention modalities for humans. The rodent cancer models have undergone constant refinement and advancement, recently in incorporating current molecular and cellular aspects of cancer formation. Initial studies by Yamagiwa and coworkers in Japan showed an association between coal tar and skin cancer using rodents. Kennaway and Hieger extracted specific chemical carcinogens from the coal tar that were responsible for the cancer induction in the rodent skin. These studies supported the earlier epidemiological observations of Pott and Butlin linking human exposure to soot and cancer. Similarly, Hueper and colleagues showed that the specific chemical components of the aniline dyes were responsible for the induction of bladder cancer in humans using a canine model. These findings correlated with the epidemiological studies by Rehn on the aniline dye workers. Berenblum and coworkers showed in a mouse skin model that the development of cancer required multiple steps and specific chemical agents could function at each of these steps; initiation and promotion. The Millers, in the 1950s showed a relationship between chemicals and their ability to bind to macromolecules (including DNA) with their carcinogenic ability. Thus these studies, using epidemiological and occupational approaches coupled with investigations in experimental laboratory models, have confirmed a linkage between exposure to carcinogenic chemicals and physical agents and human cancer induction. Table 15.2 Selected Historical Events in Chemical and Physical Carcinogenesis An understanding of the cancer process requires an understanding of the scientific terms involved in defining neoplasia (Tables 15.3 and 15.4). The terms cancer, tumor and neoplasia are often used interchangeably but in proper terminology neoplasia refers to new growth or autonomous new growth of a tissue (Table 15.3). A neoplastic lesion is referred to as a neoplasm. Neoplasms can be either benign or malignant in behavior. Benign neoplasms are lesions characterized by expansive growth, frequently exhibiting slow rates of proliferation that do not invade surrounding tissue or other organs. In contrast, a malignant neoplasm (cancer) demonstrates invasive growth characteristics, capable of spreading throughout the body. Metastases are growths in other tissues and organs that come from the cells of the primary neoplasm. A carcinogen is an agent, chemical or physical, which causes or induces neoplasia. This definition has been expanded to include an agent whose administration to previously untreated animals leads to a statistically significant increased incidence of neoplasia of one or more histogenetic types as compared with the incidence of the appropriate untreated control animals. Thus, both benign and malignant neoplasms are included in this definition of carcinogens. The term tumor describes a lesion that may or may not be neoplastic, and is characterized by swelling or an increase in size. In its truest sense, the term cancer describes those neoplasms that are malignant. Table 15.3 Definitions of Neoplasia Table 15.4 Nomenclature For classifying neoplasms, both, the tissue of origin and the characteristics of the type of tissue are incorporated into the nomenclature (Table 15.4). For benign neoplasms, the tissue in which the lesion is developed is frequently followed by the suffix “oma.” For example, a benign neoplasm from fibroblasts would be termed fibroma, and a benign neoplasm from the glandular epithelium would be termed an adenoma. Malignant neoplasms from epithelial origin are called carcinomas, while those derived from mesenchymal origin are referred to as sarcoma. Thus, a malignant neoplasm of fibrous tissue would be a fibrosarcoma, while that derived from bone would be an osteosarcoma. Similarly, a malignant neoplasm from the liver would be a hepatocellular carcinoma, while that derived from skin, referred to as a squamous cell carcinoma. Preneoplastic lesions have also been observed in a number of target organs in both animal models and humans and reflect an early reversible lesion in neoplasm progression. The characterization and study of preneoplastic cells has led to further understanding of the relationship process of cancer formation. Agents that cause cancer (carcinogens) can be chemicals, viruses, hormones, radiation, or solid-state materials. To be considered a carcinogen, the agent needs to either produce new neoplastic growth in a tissue or organ or increase the incidence and/or multiplicity of background spontaneous neoplastic formation in the target tissue. Carcinogens have been divided into two general categories based on the mechanism of action by which carcinogen functions. These have been labeled as genotoxic and nongenotoxic (or epigenetic). Genotoxic carcinogens, as the term implies, interact physically with DNA to damage or change its structure resulting in mutational event. Nongenotoxic (epigenetic) carcinogens may effect DNA expression without modifying or directly damaging DNA structure, or may create a situation in a cell or tissue that makes it more susceptible to DNA damage from other sources. Common features of genotoxic and nongenotoxic carcinogens are shown in Table 15.5. Classification of carcinogens based on mechanism has been important in developing meaningful scientifically based approaches to determining the relative human cancer risk after exposure to the cancer-causing agent. Table 15.5 Characteristics of Genotoxic and Nongenotoxic Carcinogens The majority of genotoxic, DNA reactive chemical carcinogens are found as parent compounds or procarcinogens. Procarcinogens are relatively stable chemicals that require subsequent metabolism to be carcinogenic. Metabolism occurs either in the liver or in the target tissue (where the cancer arises) itself. Since they require metabolic activation, they are referred to as indirect-acting carcinogens. A direct-acting carcinogen does not require metabolic activation. Historically, the terms procarcinogen, proximate carcinogen, and ultimate carcinogen refer to the parent compound (procarcinogen) and its metabolite forms, either intermediate (proximate carcinogen) or final (ultimate carcinogen) that reacts with DNA (Figure 15.1). The ultimate form of the carcinogen is the chemical species that produces sufficient DNA damage to induce mutation. The ultimate carcinogenic chemical forms of the most highly studied genotoxic carcinogens have been identified. Indirect-acting genotoxic carcinogens usually produce their neoplastic effects, not at the site of exposure (as seen with direct-acting genotoxic carcinogens) but at the target tissue where the metabolic activation of the chemical occurs. Examples of selected direct and indirect genotoxic carcinogens showing their pro and ultimate forms are shown in Table 15.6. Figure 15.1 Structures of indirect-acting carcinogens and their DNA reactive metabolites. The procarcinogen form (parent compound) and metabolites, the proximate (Px) and ultimate (Ut) carcinogenic forms are shown. Table 15.6 Characteristics of DNA Reactive/Genotoxic Carcinogens The reaction of a carcinogen with genomic DNA either directly or indirectly may result in DNA adduct formation or direct DNA damage, and produces a mutation if not repaired (see Chapter 14). Many ultimate carcinogens that produce mutation are alkylating electrophiles. Several mechanisms of mutagenesis from exposure to genotoxic carcinogens are known to occur. These include transitions and transversions, frame shift mutations, and DNA strand breaks as already discussed in Chapter 14. Transitions are a substitution of one pyrimidine by the other or one purine by the other, while a transversion occurs when a purine is replaced by a pyrimidine or a pyrimidine is replaced by a purine. Carcinogens can induce transitions and transversions when carcinogen adducts are formed and are misread during the DNA repair process. A shift, (frame shift mutation) may also result when the carcinogen DNA adduct is formed on a nucleotide. In a third case, DNA strand breaks can occur as a result of excision repair mechanisms that are incomplete during DNA replication. All of these events are dependent upon the location of the adducts in the genomic DNA, when in the cell cycle the adducts are formed, and the type of DNA repair enzymes that respond to the damage. While carcinogen DNA adducts can be formed at all sites in DNA, the most common sites of alkylation include the N7 of Guanine, the N3 of adenine, the N1 of adenine, the N3 of Guanine, and the O6 of guanine (Figure 15.2). Figure 15.2 DNA targets for possible adduct formation by alkylating carcinogens. Besides alkylation, another common modification to DNA is the hydroxylation of DNA bases. Oxidative DNA adducts have been identified in all four DNA bases (Figure 15.3), with 8-hydroxyguanine being the most prevalent oxidative DNA adduct formed and measured. The source of oxidative DNA damage is typically free radical reactions that occur endogenously in the cell or from exogenous sources. DNA damage and mutation induced through oxidative DNA adducts may be responsible for the “spontaneous” formation of initiated cells and tumors. Figure 15.3 Structures of selected oxidative DNA bases. Finally, modification of the methylation status of DNA has been shown to result in heritable changes in gene expression. Hypomethylation has been associated with increased transcription of genes, while hyper methylation produces a reduction of gene expression. Chemical carcinogens may inhibit DNA methylation by several mechanisms including single-strand breaks in the DNA, forming covalent adducts, alteration of methionine pools, and inactivation of the DNA methyltransferase. Although a large number of adducts can be formed following exposure to chemicals, whether a particular DNA adduct will result in mutation and participate in the carcinogenesis process is dependent in part on the persistence of the DNA adduct through the process of DNA replication, which is also in part dependent upon DNA repair. The process of carcinogenesis involves genomic mutation in a cell with subsequent cell proliferation of the mutated cell. This multistage, multistep process has been extensively study in animal models and similar steps have been shown to occur in human cancer formation. The multiple cancer process can be exemplified using both mechanisms, using the corresponding pathology to illustrate the stages. Studies with rodent as well as the pathology of human cancers have shown that the carcinogenesis process involves a series of definable and reproducible stages. Using the most basic definition, three stages, initiation, promotion, and progression, have been shown (Figure 15.4). These stages follow a temporal sequence beginning with a normal cell and completion with a neoplastic cell. The biological characteristics of these stages are shown in Table 15.7. Figure 15.4 Multistage model of carcinogenesis involving initiation, promotion, and progression stages. Table 15.7 Characteristics of Multistep Process of Carcinogenesis This initial step of the process involves the change of a normal cell to a mutated cell through a stable, heritable genetic change and is termed initiation. The initiation (mutation) becomes “fixed” (heritable) when the DNA adducts or DNA damage are not repaired completely prior to DNA synthesis. Once formed, initiated cells can remain in a nondividing state, or the mutations may be incompatible with viability of the cell and the cell dies, or the cell may proceed through additional cell divisions, resulting in the proliferation of the initiated cell. Chemical carcinogens that covalently bind to nuclear DNA and form adducts that result in mutations are initiating agents. Examples of initiating carcinogens include compounds such as polycyclic hydrocarbons and nitrosamines, biological agents, certain viruses, and physical agents such as X-rays and UV light. Many of the chemical carcinogens that function as initiators are indirect-acting genotoxic compounds requiring metabolic activation by the target cell to produce an ultimate form of the carcinogen that is then able to bind to nuclear DNA, resulting in the formation of a DNA adduct. Many compounds that work at the initiation stage have the ability to function at all stages of the cancer process and are thus complete carcinogens. It is important to note that the stage of initiation, that is, the formation of a mutated cell, can also occur spontaneously through misrepair of normally acquired DNA damage during DNA replication. The cell formed during this spontaneous initiation process might have a similar alteration of its DNA sequence to that observed with physical and chemical agents. Agents that work at the tumor initiation stage are usually genotoxic in behavior. As such their target tissue is dependent on compound metabolism and site of treatment. Frequently, tumor initiators induce tumors at multiple tissue and organ sites. A second step of the cancer process involves the selective clonal expansion of initiated cells that results in a preneoplastic lesion. This stage is often referred to as tumor promotion. Exogenous and endogenous agents that act on this stage are referred to as tumor promoters. Tumor promoters are not mutagenic and generally do not induce tumors by themselves. In general, they act through a modulation of gene expression that increases the number of cells, and these cells have been initiated either directly (via a mutagen) or indirectly (via an endogenous rate of misrepair) forming a preneoplastic lesion in the target organ. The tumor promotion stage of the cancer process requires continuous exposure to the tumor promoter. While initial exposure to a tumor-promoting agent produces a transient increase in cell growth, only the initiated cells within the tissue sustain this increase in cell proliferation and/or DNA synthesis with continual treatment to the agent. Repeated applications of the tumor-promoting compound induce a continual clonal expansion of the initiated cell population (Figure 15.4). The stage of tumor promotion is reversible whereby removal of the agent results in an increase in cell death (most likely via apoptosis) in the lesion with a return back to a single or small group of initiated cells. Carcinogens that function at the tumor-promotion stage exhibit a well-defined threshold in their dose–response pattern. For each agent, a certain dose and/or frequency of application must be achieved to produce the tumor promotion. Doses below this threshold do not modify gene expression sufficiently to produce the selective clonal expansion necessary for the development of a preneoplastic lesion. Multiple chemical compounds as well as physical agents have been linked to the tumor-promotion stage of the cancer process. Tumor promoters in general 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. The final stage of the carcinogenesis process involves the formation of the benign or malignant neoplasm. This stage is frequently referred to as the progression stage since it involves the progression from preneoplasia to neoplasm. During the progression stage, additional modification of genomic DNA and even chromosomal damage such as aberrations and translocations occurs, producing cells with the neoplastic lesion that are further independent from growth control of the body. An accumulation of nonrandom chromosomal aberrations and chromosome instability are hallmarks of progression. As noted earlier, mutations involve changes in the arrangement of the bases that comprise a gene. Two general types of mutations can occur: hereditary (germ line) mutations and acquired mutations. Hereditary mutations are defects in the genetic code, inherited by a child from its parent. Hereditary mutations are present in zygotes and, as such, are expressed in all cells of the body including sperm or eggs. As such, a hereditary mutation can be passed from generation to generation. Approximately 5–10% of human cancers are attributed to hereditary mutations. Those mutations acquired during the lifetime of the individual, usually in somatic cells, are the most frequent mutations seen in cancer accounting for 90–95% of human cancer and are induced by chemical or physical agents. Acquired mutations are found in a single cell, or the prodigy of that cell, unlike the germ line mutations. It is important to note that mutations can occur at multiple steps of the entire cancer process. Using the three-stage model noted earlier, mutation plays an important role in the initiation and progression stage of carcinogenesis. Mutations in an oncogene, tumor-suppressor gene, or other genes that control the cell cycle can result in a clonal cell population with a proliferative or survival advantage. The development of a tumor requires many such events, occurring over a long period of time, and for this reason human cancer induction often takes place within the context of chronic exposure to chemical carcinogens.
CHEMICAL CARCINOGENESIS
Exposure
Percent Human Cancer Deaths
Infection
10
Occupational
4
Medicines and medical procedures
1
Sexual behavior
7
Food additives
1
Diet
35
Alcohol
4
Tobacco
33
Pollution
2
Geophysical factors
3
15.1 BACKGROUND
Date
Investigator(s)
Finding
1775
Pott
Reported on a linkage between English chimney sweeps and scrotal cancer
1875
Thiersch
Noted a linkage between sunlight exposure and skin cancer
1879
Harting and Hesse
They reported on an association between uranium miners and the development of lung cancer
1892
Butlin
Studies with European chimney sweeps showed a distinct difference in cancer induction compared to English sweeps; he attribute this to age of workers and hygiene
1895
Rehn
Noted an increased incidence of bladder cancer in workers involved in the manufacture of aniline dyes
1902
Frieben
Proposed that exposure to X-rays was linked to cancer in humans
1915
Davis
Proposed that there was a linkage between oral cancer in pipe smokers and betel nut chewers
1928
Yamagiwa, Ichikawa, and Tsusui
Showed in an experimental rodent model, the induction of skin tumors by coal tar
1930
Kennaway and Hieger
Showed that the tumor induction of skin cancer in rodents by coal tar was the result of a specific chemical, dibenz[a,h] anthracene
1934
Wood and Gloyne
Proposed a linkage between the metals arsenicals, beryllium, and asbestos and human cancer
1938
Hueper, Wiley, and Wolfe
Extended the work of Rehn, showing that the induction of urinary cancer by aniline dyes was due to specific chemicals, in this case 2-naphthylamine
1941
Berenblum, Rous, MacKenzie, and Kidd
Showed experimentally, the two stage (initiation and promotion) model of cancer induction using the mouse skin
1951
Miller and Miller
Showed that the effects of chemical carcinogens was due to the carcinogen binding of the chemical or its metabolite to cellular macromolecules
15.2 NOMENCLATURE AND DEFINITIONS OF NEOPLASIA
Neoplasia
Neoplasm
Metastases
Tumor
Cancer
Carcinogen
Tissue of Origin
Benign Neoplasm
Malignant Neoplasm
Epithelial Tissue
Mesenchymal
15.3 CLASSFICATION OF CARCINOGENS
Genotoxic carcinogens
Nongenotoxic carcinogens
A. Direct-acting carcinogens (no metabolism needed)
B. Indirect-acting carcinogens (require metabolic activation)
15.4 MULTISTAGE CARCINOGENESIS
Initiation
Promotion
Progression
Tumor Initiation
Tumor Promotion
Tumor Progression
Mutations
DNA Repair Mechanisms
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