Because most carcinogens are mutagens, regulatory agencies often consider genotoxic mechanism as the default mechanism, unless there is sufficient evidence to indicate otherwise. Correlation studies of mutagenicity and carcino-genicity of chemicals in rodent bioassays showed a positive correlation ranging from ~70% to > 90%, depending on the database (19-21). The genotoxic mechanism undoubtedly accounts for most cancers.
In carcinogenesis, mutation may result in a gain or loss of function, depending on the role of the affected gene in cancer development. In general, activation (usually mutation) of oncogenes is considered a gain of function, for the new phenotype is the appearance of a new function. Oncogene activation often involves point mutations, chromosome translocations, and sometimes gene amplifications (22). In contrast, mutations in the tumor suppressor genes are loss of function, for which the mutated genes can no longer suppress tumor growth (7). Mutations in suppressor genes are often point mutations or deletions. Because the two copies of the tumor suppressor genes have to be inactivated for tumor expression, the term "loss of heterozygocity" (LOH) is used to denote the loss of the second copy of the allelic gene in a heterozygous genotype with a pre-existing mutation.
Mutated cells presumably acquire selective advantages, such as rapid growth rate, growth in adverse environment, and genome instability to permit further genetic alterations (23). Accumulation of genetic changes over time results in tumor cells.
The common types of genotoxic damage are described below.
A genetic change is defined as a change in the DNA sequence. A change in a single nucleotide is a point mutation. The two common types of point mutations
Genetically altered cell
Genetically altered cell
Nongenotoxic carcinogenesis fig. 1. Multistage carcinogenesis. The major difference in genotoxic and nongenotoxic carcinogenesis is at the first stage, in which genotoxic carcinogens induce mutations and nongenotoxic carcinogens create a susceptible state for genetic changes. Genetic changes occur at subsequent stages for both mechanisms.
are base-pair substitution and frameshift mutations. Base-pair substitution can be induced by a variety of chemicals including nucleoside analogs (such as bromodeoxyuridine, aminopurine) which result in mismatch of base pairs (transition or transversion) after DNA replication. It can also be induced by misrepair of DNA damage (excision or post-replicational repair) or imbalance of the nucle-otide pool (such as excessive thymidine). Frameshift mutations involve an insertion or deletion of nucleotides, which results in a change in the reading frame of the coding sequence. This type of mutation can be caused by intercalating agents (such as 9-aminoacridine, ICR-191) or by deletions (such as depurination, illegitimate recombination).
Changes in multiple nucleotides, depending on their severity, can be detected as chromosome aberrations by cytogenetic techniques. These changes involve different magnitudes of deletion, translocation, insertion, inversion, or amplification. Deletion is caused by DNA breaks, translocation, or illegitimate recombinations. Gene amplification is caused by illegitimate repetitative recombinations. It also generates a homogeneous staining region (HSR) in the chromosome and double-minute chromosomes containing the amplified DNA sequence. Changes at specific regions of the chromosome can be identified by chromosome banding (G-band, Q-band) or, if the DNA sequence and DNA probes are available, by fluorescence in situ hybridization (FISH) (24).
The genetic consequence of these changes depends on the mutation site (intron, exon) and the function of the mutated gene (structural, regulatory). These changes result in altered gene transcription, mRNA processing, or translation that causes the absence of or defective gene products.
Studies on the interactions between chemical and DNA reveal several common mechanisms of mutagenesis. Two mechanisms often discussed are DNA ad-ducts and free radical damage.
a. DNA Adducts. The term DNA adduct has been loosely ascribed to different types of DNA modifications. The discussion below is confined to the alkylated DNA adducts and macromolecular (bulky) DNA adducts. The DNA modifications by oxidative damage, sometimes also referred to as DNA adducts, are discussed in a separate section.
Certain classes of chemicals, notably alkyl sulfates, N-nitrosamines, N-nitrosoureas, and nitrogen mustards can alkylate DNA to form DNA adducts (25). The alkyl sulfates and nitrogen mustards react with DNA to form N7-alkyl-guanine and N3-alkyladenine, and the N-nitroso compounds react with oxygen to generate O6-alkylguanine and O4-alkylthymidine (26). These alkylated adducts, if not repaired timely or correctly (27), are carcinogenic. Unrepaired adducts often undergo spontaneous depurination (28), and if misrepaired (29), will result in base-pair substitutions. Another well-known mutagenic adduct is N2-3-etheno-guanine, which is induced by chloroethylene oxide, the metabolite of vinyl chloride (30). These alkylated DNA adducts can cause DNA configuration changes that interfere with DNA replication and transcription (31, 32).
A few chemicals are known to induce macromolecular DNA adducts. They are the polycyclic aromatic hydrocarbons (PAH) (such as benzo(a)pyrene) (33), mycotoxins (such as aflatoxin B) (34), chemotherapeutic agents (such as cisplati-num, 8-methoxypsoralen) (35, 36) and environmental chemicals (such as styrene oxide) (37). These bulky adducts, as expected, change DNA configuration and cause DNA damage. In fact, bulky adducts are often detected in tumor tissues and are used as biomarkers to study molecular dosimetry for exposure assessment (38, 39).
Recent studies revealed a class of bulky DNA adducts known as I-compounds. These are covalent adducts generated by endogenous reactive intermediates of oxygen metabolism. The I-compounds are classified as type I and type II. The type I compounds are species-specific adducts and their formation is affected by environmental factors. An increase of type I compound was associated with a decrease of preneoplastic lesions, which has led to the speculation that they may be protective for carcinogenesis. The type II compounds, in contrast, are generated by DNA damages or DNA cross-links. They are found in tumor tissues and are associated with tumor development (40).
b. Free Radical DNA Damage. Free radicals are reactive atoms with unpaired electrons. Radicals of the oxygen and nitrogen species are known to oxidize DNA and induce damage. Oxidative damage is associated with inflammation, neurodegenerative diseases, aging, and cancer (41-43). Acute and large amount of oxidative damage are cytotoxic to the cells and chronic low level of damage may interfere with cell cycle control (44).
The common reactive oxygen species are superoxide (O2-), hydroxyl radical (Off), peroxyl radical (RO^2), alkoxyl radical (RO^), ozone (O3), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hypochlodrite (HOCl). The reactive nitrogen species are nitric oxide (NO2) and peroxynitrite (ONOO). The reactive oxidative radicals are generated by normal chemical metabolism, and they can sometimes function as mediators of cell division and signal transduction (45-47). Chemicals such as peroxisome proliferators (48) and asbestos fibers are known to generate reactive oxidants (49)
As expected, oxidative DNA damage, if not repaired correctly (50), results in gene mutations, DNA breaks, and cross-links (51, 52). A common DNA modification involves the reaction with the hydroxyl radical (Off). Hydroxylation of DNA forms 8-hydroxydeoxyguanosine and 8-hydroxyguanine (53), but adducts of the other nucleotides were also reported (54). In addition, the Off radical can also oxidize or hydrolyze the deoxyribose moiety of DNA, generating apurinic or apyrimidinic sites susceptible to mutations or DNA breaks (52).
The nongenotoxic mechanisms of carcinogenesis do not involve DNA sequence changes at an early stage. Nongenotoxic carcinogens are defined by their absence of mutagenicity in genetic toxicology test (16-18). The nongenotoxic mechanisms are also referred to as "secondary mechanisms" (55), with the implication that they are alternate mechanisms. Unlike the genotoxic mechanism that is based simply on mutations, the nongenotoxic mechanisms are much more diverse, and in some cases, as yet speculative (56). In fact, most nongenotoxic carcinogenesis are species-, sex-, or tissue-specific, and they are often observed after high dose or prolonged exposures (57, 58). These mechanisms have been extensively reviewed (55, 59). A brief summary of the major mechanisms is included below.
In the absence of mutation, phenotypic changes can only be achieved by changes of gene expression. The most well-studied mechanism of gene regulation is methylation of DNA. DNA methylation often occurs at cytosine and is mediated by methyltransferase to form 5-methylcytosine. Hypermethylation suppresses gene expression, and conversely, hypomethylation enhances expression. Unregulated hypomethylation thus has been proposed to be a mechanism for nongenotoxic carcinogenesis (60, 61). However, hypermethylation has also been observed in carcinogenesis (62). This inconsistency may be explained by the functions of the genes that are involved, whether they have to be activated or silenced in neoplasm, such as the tumor suppressor and the senescence genes (63). Incidentally, methylation can be reversed by an inhibitor of methyltransferase, 5-aza 2'-deoxycytidine, which is commonly used in experimental studies. Recent studies showed that although methylation of DNA has always been considered a nongenotoxic process, hydrolytic deamination of 5-methylcytosine can induce mismatch point mutations and that the DNA methylation sites are often mutation hot spots in human tumors (64).
Several common nongenotoxic mechanisms of carcinogenesis are discussed below.
The rationale of cell proliferation-induced cancer is based on the concepts of spontaneous mutation rate and clonal expansion of pre-existing mutants, two different processes for the generation of mutants. In the first process, the number of mutations is a function of the mutation rate of the gene (mutation/cell/generation) and the number of cell divisions. Theoretically, with the accumulation of random mutations over many cell divisions, a mutation in the cancer gene would initiate carcinogenesis (65). The second process is clonal expansion of pre-existing mutants in the cancer gene upon cell divisions. This process is analogous to the clas sic initiation-promotion model of carcinogenesis in which an initiated cell is promoted by cell proliferation. Enhanced cell proliferation in both cases enhanced the generation of cancer cells and shortened the time for carcinogenesis. The process may appear genetic, but the first stage is stimulation of cell proliferation, not direct damage to the DNA.
Two types of cell proliferation are induced by different mechanisms. They are regenerative (or compensatory) proliferation after toxic damage to the tissue and growth factor-induced cell proliferation. Examples of these two mechanisms are described below.
a. Regenerative Cell Proliferation. Male rat renal tumor: most rodent kidney tumors are induced by genotoxic chemicals. A specific type of tumor, the renal tubule cell tumor in the male rat, has been proposed to be induced by regenerative cell proliferation as a result of chronic kidney damage (66). Unlike in other species, or even in female rats, the male rats produce a large amount of protein called a2u-globulin in the liver. Several kidney carcinogens in male rats such as d-limonene, tetrachloroethylene) are capable of binding to a2u-globulin. These bindings are believed to interfere with the degradation of a2u-globulin, which results in the accumulation of a2u-globulin in the lysosomes of renal proximal tubule cells. It is also possible, however, that undegraded a2u-globulin acts as a vector that carries the carcinogens to the kidney (67). Regardless, prolonged accumulation of a2u-globulin or a2u-globulin-chemical complexes in the kidney appears as hyaline droplets, which are toxic to the cells. Regenerative cell proliferation is stimulated which resulted in atypical tubule hyperplasia, leading to the development of renal tubule tumors (68).
Male rat urinary bladder tumor: male rats are also unique in their susceptibility to urinary bladder tumor induced by high doses of sodium salts of moderate or strong organic acids (e.g., sodium ascorbate, saccharin). These chemicals are known to form calcium phosphate precipitates in the urine at high protein concentration and the amount increases at pH above 6.5 (69-71). The precipitates are microcrystals, amorphous precipitates, or calculi that irritate the surface epithelium of the bladder. The male rats are sensitive because of their relatively high concentration of protein, such as a2u-globulin, in the urine as compared with female rats and other rodent species. Prolonged irritation of the bladder cells causes cell death, which stimulates regenerative cell proliferation and may result in bladder tumors (72).
Rodent forestomach tumor: although most forestomach tumors are induced by genotoxic chemicals, some nongenotoxic carcinogens (such as butylated hy-droxyanisole, chlorothalonil) are known to induce forestomach tumors. The mechanism proposed is chemical irritation of the forestomach epithelium followed by cell death and regenerative proliferation (73,74). Early cytotoxic changes were reportedly reversible, which indicated the epigenetic nature of the mechanism. Morphological changes are epithelial damage, hyperplasia, hyperkeratosis, dysplasia, papilloma, and eventually squamous cell carcinoma (75).
Rodent liver tumor: most halogenated hydrocarbons are rodent liver carcinogens. Several nongenotoxic agents, including chloroform, induced liver tumors in mice. A recent rodent bioassay in chloroform showed liver tumors in female mice at high doses. This observation led to the postulation that liver tumor induced by chloroform was caused by regenerative cell proliferation after toxic damage of liver cells (76). Cell death at high doses probably was caused by phosgene, a metabolite of chloroform generated by cytochrome P450 enzymes (77). Again, regenerative cell proliferation was proposed to be the cause of liver tumor induction.
b. Growth Factor-Induced Cell Proliferation. Hormones. Rodent thyroid follicular cell tumor: in contrast to human thyroid tumors that are mostly induced by ionizing radiation, thyroid follicular cell tumors in rodents are induced by genotoxic or nongenotoxic carcinogens. Several nongenotoxic chemicals (such as perchlorate, thiorueas, lithium, lupiditine) are known to perturb the balance of the thyroid stimulating hormone (TSH) in rodents. These chemicals are referred to as goitrogens and their modes of action are different. They may deplete iodine accumulation by inhibiting iodine trapping in the thyroid or by blocking binding of iodine and coupling of iodothyronine to form thyroxine and triiodothyronine. Alternately, they may inhibit thyroid hormone secretion by pro-teolysis or enhance metabolism of thyroxine by inducing liver metabolic enzymes (78). Disruption of thyroxine function activates the pituitary-thyroid feedback mechanism, which increases the production of TSH. An increase of TSH in the thyroid, if sustained, stimulates the proliferation of thyroid follicular cells through a TSH receptor-mediated signal transduction process. Excess cell proliferation may result in thyroid tumors (79, 80).
Estrogen-induced tumor: estrogen and its metabolites are known to induce mammary tumors in rodents and humans, pituitary tumors in rats, and kidney tumors in hamsters. Estrogen is metabolized by cytochrome P450 enzymes mostly in the mammary gland and uterus, which forms hydroxylated metabolites. Chemicals that alter estrogen metabolism (such as benzo(a)pyrene, dimethylbenz(a)an-thracene) are known to induce DNA synthesis, decrease apoptosis, and enhance cell proliferation (81). At least two mechanisms have been proposed, and they are not mutually exclusive. One proposal is that estrogen or its metabolites directly activates the estrogen receptors to stimulate cell proliferation. The other proposal is that the 4-hydroxylated catechol metabolites of estrogen can undergo reduction-oxidation to generate free radicals. The free radicals then react with DNA to generate 8-hydroxylguanine and other DNA modifications, resulting in genetic damage (82-85). Several estrogen-like compounds (such as diethylstilbestrol, bisphenol A, nonylphenol, polychlorinated biphenyls) are also known to be carcinogenic, but their mechanisms have not been well studied (86, 87).
Organic chemicals. Several nongenotoxic polychlorinated aromatic hydrocarbons can induce cell proliferations through a common aryl hydrocarbon (Ah) receptor, an example of which is 2,3,78-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is known to bind to the Ah receptor and initiates a signal transduction pathway that triggers cell proliferation. Enhanced cell proliferation is proposed to be the mechanism TCDD carcinogenesis (88).
Peroxisomes are membrane-bound organelles that contain oxidation enzymes. They are catalase, hydrogen peroxide-generating oxidases and a fatty acid P-oxidation enzyme system (89, 90). Several classes of chemicals (such as hypo-lipidemic drugs, phthalate esters, and phenoxy acid herbicides) are known to stimulate the proliferation of peroxisomes, which results in increases in the number and sometimes the size (volume density) of peroxisomes. The peroxisomes in rodent livers are especially susceptible to proliferation stimulation. Peroxisome proliferation in rodent livers resulted in liver cell proliferation, liver hyperplasia, hypertrophy, and neoplasm.
Concomitant to peroxisome proliferation, enzyme activities for fatty acid P-oxidation and P450 enzymes (CYP4A subfamily) in the liver are also increased. Catalase activity, however, is increased but not proportionally (89). This imbalanced increase produces high levels of hydrogen peroxide, which may cause oxidative damage to the DNA in the liver (48).
The stimulation of peroxisome proliferation by chemicals is mediated by peroxisome proliferator-activated receptors (PPAR) (91, 92). The PPAR belong to a steroid hormone receptor superfamily and are present in mice, rats, frogs, and humans. At least three subtypes of PPAR (alpha, beta, and gamma) were identified (93). Peroxisome proliferator-activated receptor alpha was found to mediate the stimulation of peroxisome proliferators in mice, and the total PPAR level increases in response to peroxisome proliferation in rats and mice (94, 95). Activation of PPAR induces the transcription of the liver genes for fatty acid metabolism (94-96). Rats and mice are more susceptible to peroxisome proliferators than hamsters, guinea pigs, and monkeys.
It is important to note that the amount of PPAR alpha in humans is much less than in rodents (97, 98). Studies in humans treated with hypolipidemic drugs (rodent peroxisome proliferators) and in cultured human hepatocytes treated with peroxisome proliferators did not show peroxisome proliferation (92, 99, 100). The role of peroxisome proliferation in human carcinogenesis requires further evaluation. At present, none of the known human liver carcinogens (IARC group 1 carcinogens) is a peroxisome proliferator (101, 102). Rodent peroxisome pro-liferators do not appear to pose a cancer risk to humans.
A defect in the intercellular communication has been proposed to be an epi-genetic mechanism of carcinogenesis (103-106). Intercellular communication is mediated through gap junctions, which are membrane structures that allow the transfer of small molecules between cells. The molecules can be nutrition or signal transduction molecules. A gap junction protein, connexin, was identified as an essential protein for cell communication. Mislocation of connexin in the membrane was observed in tumor cells (104). The expression of the connexin gene was also down regulated in tumor cells, and inactivation was caused by hypermethyla-tion (107). The connexin gene appears to act as a tumor suppressor gene (that is, functional in normal cells), for its inactivation resulted in carcinogenesis (103, 107).
Genomic imprinting is an epigenetic modification of a parental allele of a gene in the gamete or zygote, resulting in differential expression of the paternal and maternal alleles in the offspring. This unequal expression is a departure from Men-delian genetics, but it is frequently observed in mammalian genetics. Alternations of the imprinted genes have been observed in many human cancer cells (such as Wilms' tumor, hepatoblastoma, rhabdomyosarcoma, Ewing's sarcoma) (108-110). This change is referred to as loss of imprinting (LOI). A possible consequence of LOI, as related to carcinogenesis, is the activation of an inactivated cancer gene or the inactivation of a tumor suppressor gene. The mechanism of imprinting is DNA methylation, because abnormal methylation patterns are observed in LOI regions. Moreover, studies with 5-aza 2'-deoxycytidine, an inhibitor of methylation, and in methyltransferase-deficient mice indicated that methylation is responsible for imprinting (109, 111). The most carefully studied gene for imprinting is the insulin-like growth factor 2 gene (IGF2). This gene is located on human chromosome 11p15.5, for which LOI was observed in a variety of human cancers, including kidney, bladder, cervical, prostate, testicular, and ovarian (112). In addition to human medical conditions, environmental agents have also been reported to induce LOI (113).
Was this article helpful?
Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.