Q Assignment is about Cancer is a disease of cellular mutation, proliferation, and aberrant cell growth. Home, - Cancer 1. INTRODUCTION 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. Evidence that chemicals can induce cancer in humans has been accumulating since the sixteenth century. Cancer is often perceived as largely due to inherited defective genes. However, the biggest cause of human cancer is exposure to chemicals and indeed, the biggest contributors to the burden of human cancer are chemicals contained within tobacco smoke, the diet and the environment. Chemical carcinogenesis is a multistage process in which normal cells become first initiated, then malignant and invasive. Carcinogens can be chemicals, viruses, hormones, radiation, or solid ma terials. Carcinogens may be genotoxic, meantime that they interact physi cally with DNA to damage or change its structure. Other carcinogens may change how DNA express information without modifying or directly damaging its structure, or may create a situation in a cell or tissue that makes it mo re susceptible to DNA damage from other sources. Chemicals belonging to this latter category are referred to as nongenotoxic carcinogens. Humans are constantly bombarded with physical, chemical and biological insults such as ultra-violet (UV) radiation, toxic chemicals and pathogens that can damage DNA, activate oncogenic pathways and cause inflammation. These damages can lead to precancerous lesions that can become malignant. 2. Classification of Chemical Carcinogens Carcinogens can be categorized as either genotoxic or non-genotoxic according to their specific pathogenic mechanism. Most genotoxic carcinogens are electrophiles that interact directly with DNA through the formation of covalent bonds, resulting in DNA-carcinogen complexes (DNA adducts). These complexes lead to various types of DNA damage, including the formation of cross-links between the two helices, chemical bonds between adjacent bases, removal of DNA bases (hydration) and cleavage of the DNA strands, all of which result in modifications to the information stored within the DNA. Such mutations are typically fixed by DNA repair mechanisms; however, if DNA replication occurs prior to the action of a repair mechanism, mutations can become permanent and may eventually cause tumors. Conversely, non genotoxic carcinogens have no direct interaction with DNA; they are believed to cause tumors by disrupting cellular structures and by changing the rate of either cell proliferation or of processes that increase the risk of genetic error. 3.1 Initiation The first stage of the cancer process involves initiation, a process that is defined as a stable, heritable change. Initiation is a fast, irreversible phenomenon and is transmitted to daughter cells. Initiation is an additive process, neoplastic development depends on the carcinogenic dose, increasing the dose increases the incidence and the multiplicity of resultant neoplasias and reduces the latent period of its manifestation. Chemical and physical agents that function at this stage are referred to as initiators or initiating agents. Chemicals that initiate carcinogenesis are extremely diverse in structure and include both natural and synthetic products. They are: (a) direct acting compounds, which do not require chemical transformation and (b) indirect acting compounds or pro carcinogens, which require metabolic conversion to produce ultimate carcinogens. Chemical carcinogens that covalently bind to DNA and form adducts that result in mutations are initiating agents. Included among chemicals classified as initiating carcinogens are compounds such as polycyclic hydrocarbons and nitrosamines, biological agents such as viruses, and physical agents such as X-rays and UV light. Most chemical carcinogens that function at the initiation stage of the cance r process are indirect acting genotoxic compounds that require metab olic activation in the target cell to produce the DNA-damaging event. Initiation involves a nonlethal and inheritable mutation in cells by interaction of a chemical with DNA. The activation of the carcinogen to an electrophilic DNA-damaging moiety is a necessary step for this stage. Reactive Oxygen Species (ROS) are believed to mediate the activation of such carcinogens through hydroperoxide-dependent oxidation that can be mediated by peroxyl radicals. This occurs with aflatoxin B, aromatic amines and polycyclic aromatic hydrocarbon dihydrodiols. ROS or their byproduct of lipid peroxidation, can also directly react with DNA to form oxidative DNA adducts. The presence of carcinogen-DNA adducts and oxidative DNA adducts generated by chemical carcinogens suggest an interactive role of ROS in initiation. The formation of oxidative stress may result in damage to critical cellular macromolecules including DNA, lipids, and proteins. Oxidative DNA damage may participate in ROS-induced carcinogenesis. A common form of damage is the formation of hydroxylated bases of DNA, which are considered an important event in chemical carcinogenesis. This adduct formation interferes with normal cell growth by causing genetic mutations and altering normal gene transcription. 3.2 Promotion Once the initiated cell has been encouraged to replicate, the initiated genetic damage is irreversibly fixed. When the initiated cell is subjected to promotional influences, the effect is to encourage clonal expansion of the initiated cell. A key feature of the promotion response is its reversibility, that is, if the promoting agent is removed, the initiated cell population is no longer encouraged to proliferate. The promotion stage includes epigenetic processes responsible for the formation of the malignant phenotype and survival of tumor cells. The carcinogenicity of some chemicals is augmented by subsequent administration of promoters (such as phorbol esters, hormones, phenols and drugs) that by themselves are non-tumorigenic. Application of promoters leads to proliferation and clonal expansion of initiated (mutated) cells. Promoter compounds do not interact directly with DNA and unchain biological effects without being metabolically activated. These agents increase cell proliferation in susceptible tissues, contribute towards fixing mutations, enhance alterations in genetic expression and cause changes in cellular growth control. The promoters may indirectly damage DNA by oxidation. Pathologically this results in the formation of the preneoplastic lesion. ROS generation is related to P450 enzyme activity, oxidative stress may have an important role in the clonal expansion of the initiated cells. 3.3 Progression As the initiated cell population is expanded during promotion, individual cells acquire further genetic damage, which can be either agent mediated or spontaneous, thereby, introducing genetic heterogeneity into the promoted population. Acquisition of these changes is irreversible and progressive. Progression consists of production and selection of cell clones able to compete favorably with the normal cell environment. In progression, a neoplastic phenotype is acquired through genetic and epigenetic mechanisms. During progression, cell proliferation is independent from the presence of stimulus. It is characterized by irreversibility, genetic instability, faster growth, invasion, metastization and changes in the biochemical, metabolic and morphological characteristics of cells. 4. Metabolic Activation of Chemical Carcinogens Most of the reactions of carcinogens with cellular macromolecules resulted from covalent bond formation between an electrophilic form of the carcinogen and the nucleophilic sites in proteins and nucleic acids 4.1 Donors of Simple Alkyl Groups Included in this group are the dialkylnitrosamines, dialkylhydrazines, aryldialkyltriasenes, alkylnitrosamides, and alkylnitrosimides. The alkylnitrosamides and alkylnitrosimides do not require enzymatic activation because they can react directly with water or cellular nucleophilic groups. The alkylnitrosamines, alkylhydrazines, and alk yltriazenes, however, undergo an enzyme mediated activation step to form the reactive electrophile. These agents are metabolically dealkylated by the mixed-function oxidase system in the microsomal fraction (endoplasmic reticulum) of cells, primarily liver cells. The monoalkyl derivatives then undergo a nonenzymatic, spontaneous conversion to monoalkyldiazonium ions that donate an alkyl to cellular nucleophilic groups in DNA, RNA, and protein. 4.2 Cytochrome P- 450 Mediated Activation A number of carcinogenic chemicals are chemically inert nucleophilic agents until they are converted to active nucleophiles by the cytochrome p-450–dependent mixed function oxidases. A wide variety of chemical carcinogens such as aromatic and heterocyclic amines, aminoazo dyes, polycyclic aromatic hydrocarbons, N-nitrosamines, and halogenated olefins are activated by one or more of these CYPs. Some ofthese compounds are further activated by subsequent steps; for example, 2-acetylaminofluorene (AAF) is further modified by a sulfotransferase to form the ultimate DNA-binding moiety. 4.3 2- Acetylaminofluorene In1960, it was shown that AAF is converted to a more potent carcinogen, N-hydroxy AAF, after the parent compound was fed to rats. Although both AAF and N-hydroxyl-AAF are carcinogenic in vivo, neither compound reacted in vitro with nucleic acids or proteins, suggesting that the ultimate carcinogen was another, as-yet unidentified metabolite. The enzymatic reactions are involved in the conversion of AAF to carcinogenic metabolites, especially in non hepatic tissues, which oft en have low sulfotransferase activity for N hydroxy- AAF. The acetyltransferase-mediated activity converts N-hydroxy-AAF to N-acetoxy- 2 aminofluorene, which is also a strong electrophile and ma y be the ultimate carcinogen in non hepatic tissues. 4.4 Other Aromatic Amines Electrophilic forms of the aromatic amines result from their metabolic activation, and the positively charged nitrenium ion formed from naphthylamine and aminobiphenyl compounds has been implicated as the ultimate urinary bladder carcinogen in dogs and humans. Hydroxylamine derivatives of these compounds are formed in the liver and then converted to a glucuronide. The glucuronide conjugate is excreted in the urine, where the acid pH can convert it back to hydroxylamine and subsequently to a protonated hydroxylamine, which rearranges to form a nitrenium ion by a loss of water. The electrophilic nitrenium ion can then react with nucleophilic targets in the urinary bladder epithelium. 5. DNA Adduct formation Since most chemical carcinogens react with DNA and are mutagenic, interactions with DNA have been viewed as the most important reactions of these agents with cellular macromolecules. Reaction of chemical carcinogens with DNA is the simplest mechanism that explains the induction of a heritable change in a cell leading to malignant transformation; thus many investigators view this as the most plausible mechanism forinitiation of carcinogenesis. Representative agents from virtually all classes of chemical carcinogens have been shown to affect DNA in some way, and a number of distinct biochemical-reaction products have been identified after treatment of cells in vivo or in culture with carcinogenic agents. The potential biological consequences of DNA base–adduct formation by chemical carcinogens are several. In some cases, it may stabilize an intercalation reaction in which the flat planar rings of a polycyclic hydrocarbon are inserted between the stacked bases of double helical DNA and distort the helix, leading to a frame-shift mutation during DNA replication past the point of the intercalation. Many of the base adducts formed by carcinogens involve modifications of N-3 or N-7 positions on purines that induce an instability in the glucosidic bond between the purine base and deoxyribose, resulting in loss of the base and creation of an apurinic site in DNA. DNA in transcribing regions associated with the nuclear matrix also appears to be a preferential target for carcinogen binding. 6. Mechanism of Action of Chemical Carcinogens 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. Both these events can be induced or acted upon by chemical carcinogens. Chemicals that induce cancer have been broadly classified into one of two categories—genotoxic or DNA reactive, and non genotoxic or epigenetic carcinogens— based on their relative abilities to interact with genomic DNA. 6.1 Genotoxic Carcinogens Genotoxic carcinogens initiate tumors by producing DNA damage. Coal tar carcinogens including benzo(a)pyrene, pesticides such as 2-acetylaminofluorine, and azo dyes such as diamino benzamide were among the first chemical carcinogens to be studied. 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 metabolicactivation (i.e.,indirect acting carcinogens com pounds that require metabolism in order to react with DNA). Direct-Acting (Activation-Independent) Carcinogens: A variety of carcinogens do not require metabolic activation or chem ical modification to induce cancer, and are termed direct acting or act ivation independent carcinogens. These chemicals are also defined as ultimate carcinogens. Examination of the chemical structure of these agents reveals that they are highly reactive electrophilic molecules that can interact with and bind to nucleophiles, such as cellular macromolecules including DNA. The relative carcinogenic strength of direct acting carcinogens depen ds in part on the relative rates of interaction between the chemical an d genomic DNA as well as competing reactions with the chemical and other cellular nucleophiles. The relative carcinogenic activity of direct-acting carcinogens is dependent upon such competing reactions and also on detoxification reactions. 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. A number of direct-acting alkylating agents, including a number of chemotherapeutic chemicals, are carcinogenic in humans. Indirect-Acting Genotoxic Carcinogens: Many carcinogens are not intrinsically carcinogenic, but require metabolic activation to be carcinogenic. The majority of DNA reactive carcinogens are found as parent compounds, orprocarcinogens. Proca rcinogens are stable chemicals that require subsequent metabolism to be carcinogenic. 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. Indirect-acting genotoxic carcinogens include the polycyclic aromatic hydrocarbons (PAHs), nitrosamines, aromatic amines, and aflatoxin B1. 6.2 Mutagenesis The reaction of a carcinogen with genomic DNA, either directly or indirectly, may result in DNA adduct formation or DNA damage, and frequently produces a mutation. Several mechanisms of mutagenesis are known to occur. Modification of DNA by electrophilic carcinogens can lead to a number of products. Modified DNA are dependent upon 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. Transitions are a substitution of one pyrimidine by the other, or one purine by the other (changes within a chemical class), whereas a transversion occurs when a purine is replaced by a pyrimidine, or a pyrimidine is replaced by a purine (changes across a chemical class). Carcinogens can induce transitions and transversions several ways. In one scenario, when adducts (or apurinic or apyrmidinic sites) are encountered by the DNA replication processes, they may be misread. The polymerase may preferentially insert an adenine (A) in response to a noninformative site. Thus, the daughter strand of an A, C, G or T adduct will have an adenine (A) and this change is fixed (mutation) and resistant to subsequent DNA repair. A second outcome, a shift in there adding frame (resulting in a frame shift mut ation) may also result from carcinogen–DNA adducts formation. Most frameshift mutations are deletions and occur more frequently when the carcinogen–DNA adduct is formed on a nucleotide. In a third scenario, DNA strand breaks can also result from carcinogen DNA adducts. These may arise either as a result of excision–repair mechanisms that are incomplete during DNA replication or via direct alkylation of the phosphodiester backbone leading to backbone cleavage. Strand scission can lead to double-strand breaks, recombination, or loss of heterozygosity. 6.3 Damage by Alkylating Electrophiles Most chemical carcinogens require metabolic activation to exert a carcinogenic effect. The ultimate carcinogenic forms of these chemicals are frequently strong electrophiles that can readily form covalent adducts with nucleophilic targets. Because of their unpaired electrons, S, O and N atoms are nucleophilic targets of many electro philes. The extent of adducts formed is limited by the structure of D NA, where bulky electrophilic chemicals can bind, and size of the ultimate carcinogenic form. In general, the stronger electrophiles display a greater range of nucleophilic targets (i.e., they can attack weak and strong nucleophiles), where as weak electrophiles are onlycapable of alkylating strong nucleophiles (e.g. S atoms in amino acid s). In addition, the metabolic capability of a target cell will dictate the extent and types of electrophiles generated from the procarcinogenic parent. Alkylations of phosphate may occur at a high frequency. Different electrophilic carcinogens will often display different prefer ences for nucleophilic sites in DNA and different spectra of damage. Dimethylnitrosamine and diethylnitrosamine, for example, are metabolized by P450 oxidation to yield a methyl carbonium ion and an ethyl carbonium ion, respectively. Another common modification to DNA is the hydroxylation of DNA bases. Oxidative DNA adducts have been identified in all four DNA bases; however, 8-hydroxyguanine is among the most prevalent oxidative DNA adduct. The source of oxidative DNA damage is typically formed from free radical reactions that occur endogenously in the cell or from exogenous sources 7. Conclusion Many hundreds of chemicals are known to induce cancer in experimental animals but the number for which there is good evidence for cancer causation in man is relatively few. These include asbestos, various metals, aromatic amines, coal tar products and mustard gas. Among chemicals known to induce tumours in experimental animals are polycyclic aromatic hydrocarbons, aromatic amines, nitrofurans and some hydrazine derivatives. Among the large number of carcinogenic nitroso compounds only one, N-nitroso-n-butylurea has been found to induce tumours in animals. A number of models for the study of carcinogenesis have been developed over the years. There are several genes which intervene in carcinogenesis- their identification revolutionised chemical carcinogenesis and oncology. Out of all of these, protooncogenes, tumour suppressor genes and cell cycle regulator genes assume a particular importance.DNA is the primary target for chemical carcinogens is fairly well established but there is no single or unique alteration that can be associated with initiation of chemical carcinogenesis. It should be emphasized that carcinogen-induced changes in DNA do not necessarily lead to initiation because most types of DNA damage can be repaired by cellular enzymes. The presence of certain types of DNA damage in human tumors can provide molecular clues to their causation. This is exemplified by the study of mutations in RAS and p53 genes. Each carcinogen produces a molecular ‘fingerprint’ that can link specific chemicals with their mutational effects. The tumor suppressor proteins p53; p21 and Retinooblastoma protein (pRb) play crucial roles in cellular protection, because they encourage the blocking of cells at G1 phase of cell cycle. The loss of pRb protein function provokes an increase in the cell proliferation rate and an absence of terminal differentiation. p53 can interrupt the cell cycle at G1 and go on to repair DNA damage.