In this article we will discuss about:- 1. Introduction to Environmental Carcinogenesis 2. Historical Background of Environmental Carcinogenesis 3. Environmental Pollution and Carcinogens 4. Types of Carcinogens 5. Mechanism of Chemical Carcinogenesis 6. Molecular Mechanism 7. Discovery of Viral and Cellular Oncogenes 8. Discovery of Viral and Cellular Oncogenes 9. Carcinogenic Risk Assessment.
Contents:
- Introduction to Environmental Carcinogenesis
- Historical Background of Environmental Carcinogenesis
- Environmental Pollution and Carcinogens
- Types of Carcinogens
- Mechanism of Chemical Carcinogenesis
- Molecular Mechanism of Carcinogenesis
- Discovery of Viral and Cellular Oncogenes
- Discovery of Viral and Cellular Oncogenes
- Carcinogenic Risk Assessment
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1. Introduction to Environmental Carcinogenesis:
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It has been established now that a high proportion of human cancers are caused by environmental agents – mainly environmental chemicals, but also viral and physical agents. It is unclear to what extent genetic factors may contribute to individual susceptibility to chemically induced carcinogenesis. However, perhaps three-fourths of human cancers are due to environmental chemicals.
In a series of reviews of the carcinogenicity of approximately 300 substances by International Agency for Research on Cancer, 21 substances are listed as carcinogenic in humans and an additional 150 as carcinogenic in experimental animals. Others have claimed that 30 identified compounds are definite human carcinogens.
Much of the current research is directed toward the identification of carcinogenic agents among environmental substances, industrial chemicals, and drugs and the development of methodology to predict the carcinogenicity of a tested substance.
The distribution of potential carcinogens in the environment is ubiquitous. Water may contain carbon tetrachloride and other chlorinated compounds or metallic salts that may be potentially carcinogenic. Laboratory and industrial solvents such as benzene and carbon tetrachloride may also be carcinogens. Alternatively, during the cooking of food, the nitrites can react with the amines to yield carcinogenic nitrosamines.
Approximately three-fourths of the total 100-120 nitroso compounds tested are carcinogenic to experimental animals. Certain foods may also be contaminated with the aflatoxins, potentially carcinogenic compounds produced by some Aspergillus strains.
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Epidemiological investigations in man have suggested an association of increased incidence of hepatic tumors with increased dietary contamination by aflatoxins. Various polynuclear aromatic hydrocarbons, such as the carcinogen benzopyrene formed in the combustion of organic substances are potential carcinogens.
2. Historical Background
of Environmental Carcinogenesis:
The environmental basis of carcinogenesis was first put forth by an English Surgeon, Percivall Pott (1775) when he observed high incidence of scrotal cancer amongst chimney sweeps exposed to soot. It is, however, now known that soot contains a group of a potentially hazardous carcinogenic compounds termed as polyaromatic hydrocarbons.
During the 16th century it was also realized that miners were frequent victims of what is recognized as lung cancer, which was rare in the rest of the population. In the latter half of 19th century ‘coal tar’ derivatives were found to induce carcinoma of skin and vulva. Later on, Japanese scientists Yamagiwa and Ichikawa successfully induced papilomas by painting rabbit ear with ‘tar’. Kennaway (1930) induced tumors in mouse skin with a polyaromatic hydrocarbon benzo(a)pyrene.
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3. Environmental Pollution and Carcinogens
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In an urge for providing sufficient food and better life to its citizens, every country undertook industrial expansion alongwith large scale use of pesticides and chemical fertilizers which resulted in environmental pollution. Moreover, introduction of heavy metals, polyaromatic hydrocarbons, noxious gases, synthetic chemicals, dyes, food additives and a host of other pollutants also caused pollution in the wake of heavy industrialization, modernization and changed life style.
As a result of all these activities our environment was polluted by a wide variety of chemicals. There are approximately more than 700,000 chemicals in the environment and to these 1000 to 2000 new chemicals are being added every year.
Cancer has become one of the leading causes of death in many parts of the world. The exact cause of human cancer is not well understood, however, some of the aetiological agents of environmental origin (environmental carcinogens) include polyaromatic hydrocarbons, tobacco, some metals, mycotoxins, certain pesticides and drugs etc. It is now well documented that 90% of human cancers have environmental origin.
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Epidemiological surveys have revealed that many cancers originate due to occupational exposure of workers to carcinogenic pollutants. Examples include high incidence of lung cancer among workers exposed to asbestos dust and coke oven emission, liver angiosarcoma among workers exposed to vinyl chloride, bladder cancer in aniline dye workers and many others.
Various epidemiological surveys and experimental studies have shown that most of the environmental carcinogens can cause immunotoxicity resulting in altered immune function of the exposed individual developing thereby a state of either partial or complete immunosuppression. The prolonged immunosuppression makes an individual extremely susceptible to various infections and even may predispose the host to develop cancer.
4. Types of Carcinogens
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These are in general, synthetic chemicals developed primarily for laboratory research or those used in industry. Added to these are the anticancer drugs. These carcinogens do not require the host metabolic activation by mixed function oxidases (MFOs) or other enzymatic activation in generating key reactive metabolites.
To name a few of them are- p-propionlactone, 1,2,3,4-diepoxybutane, ethyleneimine, dimethyl sulphate, bis (2-chloroethyl) sulphide (mustard gas), nitrogen mustard, melphalan (sacrolysin), 2-naphthyl amine mustard (chlomaphazine), bis (chloromethyl) ether, benzyl chloride and dimethyl carbamyl chloride.
The parent compounds of this class of carcinogens are mostly inert in the microsomal fraction of liver. This system of enzymes contains CYP450(s). They require metabolic activation by mixed function oxidases (MFO). It is now known that there are at least six forms of cytochrome P-450 with different steroids and biphenyl substrates. The various forms of cytochrome P-450 show preferential hydroxylation at specific positions.
These are agents which increase the effect of directly acting carcinogens and/or pro carcinogens e.g. dilute solution of croton oil greatly enhances the effect of PAH (3-methyl cholanthrene) in causing skin tumors in mice. Tobacco tar and tobacco smoke are the co-carcinogenes that contain strong co-carcinogenic agents.
5. Mechanism of Chemical Carcinogenesis
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There is little doubt nowadays that carcinogenesis in general and chemical carcinogenesis specifically is usually a multistage process, comprising different stages from the conversion of a normal somatic to a tumor cell (transformation) and ultimately, after a long latency period, to a clinically manifest malignant tumor.
While many of the later stages of carcinogenesis (promotion and progression) are little understood at present and are fields of intensive research, especially from a biochemical and molecular-biological point of view, the stage of initiation (i.e. the transformation of a normal to a potential tumor cell) has been extensively studied in the past two decades and has finally resulted in a theory of chemical carcinogenesis that has been and is widely accepted. Some theories of initiation, especially the one by Miller and Miller is based on the concept of somatic mutation as the first step in carcinogenesis.
Any theory of carcinogenesis has to incorporate the fact that the initiation process leads to irreversible changes at the cellular level; thus, mechanistic theories must explain that the induced effect becomes permanent. The only plausible explanation at present is that the initiation step is a genetic change, probably a somatic mutation. Therefore, the direct interaction of the carcinogen or its activated metabolite/s with DNA as a promutational step has received much attention.
However, it should not be forgotten that reasonable models do exist, which discuss macromolecular cellular targets other than DNA. Thus, chemical carcinogenesis might also result from a primary alteration of a cellular RNA, since Temin has shown that RNA’s can be transcribed intracellularly as DNA and the resulting DNA can be integrated into the host genome. Dickson and Robertson discuss a mechanism of the potential regulatory role of specific RNA’s in cellular development.
Alterations of specific proteins could also have a potential for permitting the development of cellular clones with altered genomes. A possible mechanism is the greatly increased error rates of certain DNA polymerases. Another possible molecular mechanism of carcinogenesis, not involving direct genomic change in the cells, is based on the repressor-depressor systems that control the expression of genomes of viruses or bacteria.
Monod and Jacob (1962) and Pitot and Heidelberger (1963) both have argued that loss or modification of protein repressors or parts of the genome that are expressed, could result in near stable states of cellular differentiation.
This historical “review” however should by no means distract from the fact that direct interaction of chemical carcinogens with DNA and the ensuing mutation is no doubt the most probable as well as the most plausible mechanism of initiation.
The groups Lawley (1957) and Brookes (1960) first showed the reaction of direct alkylating carcinogens with nucleic acids and proteins, with covalent binding to these substrates. They also showed that DNA interaction correlated best with carcinogenicity data.
The indirectly acting carcinogen 4-dimethyl-aminobenzene (butter yellow) was first shown by Miller and Miller (1947) to bind to proteins in 1947 and, later, by the same group, also to form adducts with nucleic acids.
Identification of formed adducts invariably showed that cellular nucleophiles were the sites to which binding occurred, thus indicating that reactive intermediates of carcinogens were electrophilic chemical species.
i. The Miller and Miller Theory:
These and similar results thus formed the basis of a more generally accepted theory of chemical carcinogenesis by Miller and Miller (1966, 1970; 1977). It postulates that chemical carcinogens are either electrophiles (directly acting agents) or form electrophilic reactive metabolites in vivo from per se chemically nonreactive carcinogens (so-called procarcinogens).
The reactive electrophilic species then interacts with cellular nucleophiles, especially in DNA. DNA adduct formation is then considered as a promutagenic step which after cell replication, may lead to a mutation and therefore to a “fixation” of the change. DNA interacting carcinogens are therefore also called genotoxic carcinogens, they are usually mutagenic in appropriate test systems.
To summarize- genotoxic carcinogens bind covalently to DNA and tumor initiation is therefore the consequence of mutation(s) resulting from such interactions. This theory, based on new scientific results of carcinogenesis research, also lead to a revival of the somatic mutation hypothesis, first suggested by Boveri (1919) in 1914 and later emphasized again by K.H. Bauer (1949).
In a critical discussion of the theory, of course, it must be strongly emphasized that it explains plausibly many of the known facts in carcino-genesis, but that it is an idealized scheme that does not explain all facts. It effectively explains the initiation mechanism of genotoxic carcinogens, but it certainly cannot easily explain carcinogenesis with proven human carcinogens, such as inorganic carcinogens (arsenic, nickel, chromium) and fibrous inorganics such as asbestos.
Hormonal agents such as 17-a-estradiol or diethylstilbestrol are non-mutagenic, but carcinogenic in animal bio-assays. Similarly many other chemical compounds are carcinogenic in certain animal bioassay systems, but are non-mutagenic and probably do not interact with DNA directly.
It is now clearly evident that the title “Carcinogens are Mutagens” in the paper by B. Ames (1973) is wishful thinking and in this generalized form wrong. However, some carcinogens formally do not fit into Miller’s scheme.
Arsenicals, some nickel and chromium compounds are recognized human carcinogens and have also induced cancer in animal bioassays. The usual short-term tests for assaying mutagenicity usually give negative results with inorganic compounds; however mammalian cell systems are better suited to test for DNA and chromosomal damages- Carcinogenic metal compounds sometimes but not always induce chromosomal aberrations in different test systems such as human lymphocytes or in Chinese hamster embryo cells where they also induce transformations.
Asbestos and similar fibers, also negative in simple mutation test systems, induce chromosomal damage and also transform Syrian hamster embryo cells.
Thus for inorganic carcinogens there is some, but often not sufficient evidence tor genotoxicity in a broad sense but they probably do not form DNA- adducts; their DNA interaction, mechanistically not understood, is of a more complex nature.
6. Molecular Mechanism of Carcinogenesis:
Recent researches oriented to investigate the mechanism of carcinogenesis reveal that alterations in genes of normal cells are responsible for initiation of carcinogenesis and cancer is genetic in origin. It is now known that ‘tumor producing genes’ or ‘oncogenes’ are not only present on genetic locus of retroviruses but also in normal cells-cellular oncogenes (c-oncogenes).
Various environmental carcinogens including radiation can activate or cause enhanced expression of c-oncogenes, resulting in the formation of ‘oncogene encoded proteins’ in large amounts. Several carcinogens lead to initiation of carcinogenesis by activating c-oncogenes through deletion and/or substitution mutational mechanism.
A good amount of evidence is now available, which indicates that carcinogens can cause substitution at specific focus of c-ras oncogene leading to initiation of carcinogenesis. Weinstein (1988) has documented the role of ‘Suppressor genes’ (S-genes) in the initiation of carcinogenesis. In a normal cell S-genes control and regulate the cellular growth. The activation of c-oncogenes by carcinogens or radiation decreases the activity of S-genes.
The elimination or lesser expression of S-genes results in the removal of normal constraint on cell growth, and such genetically depleted cells, grow uncontrolled and become wild or malignant. The involvement of S-genes in carcinogenesis is evident from the experimental observations of Weinstein (1988) who noticed that the deletion of S-gene-Rb gene (located on 13th human chromosome) results in the conversion of normal retinoblastomas.
Carcinogens/radiation may cause activation of oncogenes which can disturb signal transduction responsible for normal growth and differentiation involving protein kinase C (PKC). PKC is a membrane bound enzyme often associated with regulatory functions and signal transduction for cellular growth and differentiation. The activation of c-oncogenes alongwith other events leading to carcinogenesis and tumor development are shown in Fig 17.2.
Oncogenes encode several proteins, many of which are now characterized. ‘Src’, oncogene encodes a protein kinase having mol. wt. 60,000 daltons. Several oncogene encoded proteins are produced following activation of ‘fms’, ‘raf’, ‘ras’ and ‘mos’ oncogenes and have unique property of phosphorylating cell membrane proteins and assist in bringing about carcinogenic transformation.
In the past 25 years a major revolution has occurred in understanding the molecular basis of cancer. The long drive to understand how normal cells grow and contribute to the ordered development of an organism and the nature of genetic events that disrupt this ordered process and result in cancer has become a major focus of biological research.
The explosions of knowledge from basic research on cancer has unified several disciplines of science and led to a clear understanding of the mechanisms associated with normal cell growth, genetic events that produce defects in regulatory mechanisms that lead to aberrant growth and differentiation, and finally the molecular basis for disease processes, such as cancer.
The most important breakthrough that led to this scientific revolution was the discovery that a small set of mammalian genes (probably about 100) and avian genes (approximately 200,000) controls normal development of the organism. Aberrations introduced into these genes during the life span of an organism by viral infection or by exposure to chemical carcinogens leads to cancer.
It is becoming very clear that a precise understanding of the structure of these genes and the mechanisms associated with their function will provide us with approaches to treat diseases, such as cancer, and also to correct genetically inherited forms of developmental defects. Therefore, we are at one of those exciting moments in science when many disciplines of scientific endeavor come together, and much that seemed impossibly obscure suddenly becomes clear.
It has been recognized for almost a century that cancer is a multistep process, which takes decades to develop. Because cancer cells grow at a much faster rate than normal cells and do not obey rules of ordered growth immediately suggested that cancer is an aberration of cell growth.
This observation, combined with the fact that cancer is a multistep process, suggested that involvement of multiple genetic events of the growth deregulation seen in cancer cells. Research during the past decade has shown that the genetic events include activating a group of genes, termed “oncogenes”, and inactivating another group of genes named “growth suppressor genes”.
By definition, oncogenes are genes that promote cell growth, and growth suppressor genes are those that block cell growth. A fine balance between the activities of these two groups of gene products dictates normal cell growth, and disrupting this balance provides a cell with a growth advantage that ultimately results in a neoplastic state.
7. Discovery of Viral and Cellular Oncogenes
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Oncogenes were initially discovered by studying transforming retroviruses that produce tumors in animals. Several decades ago, it was observed that chickens die predominantly of cancer, suggesting that these animals have a genetic predisposition to this disease. This observation led to examining the tumors derived from these animals for a disease transmitting agent, which led to the identification of retroviruses.
In 1991 Peyton Rous at the Rockefeller Institute in New York first isolated a retrovirus from a spontaneous chicken sarcoma. He established the viral etiology of the tumor by demonstrating that extracts derived from the tumor could be filtered through membranes that retain bacteria and nevertheless induce cancer in animals injected with the filtered extracts.
The virus isolated by Rous has been named the Rous sarcoma virus in honor of its discoverer. Most interestingly, it was found that these viruses contain RNA as their genetic material and hence was called “RNA tumor viruses” or “retroviruses”. Later studies revealed that extracts from animal tumors often contain two types of RNA tumor viruses, which were termed acute transforming viruses and leukemia viruses. These viruses differ from each other in a number of properties.
The most important biological difference between these two classes of viruses is their ability to induce tumors in vivo as a function of time. Although acute transforming viruses induce tumors in susceptible, hosts in a very short time (1 to 2 weeks), the leukemia viruses require several months to years.
A second important difference between the two classes of viruses is their ability to transform cells in tissue culture. The acute transforming viruses readily transform cells in tissue culture, whereas the leukemia viruses fail to do so. Finally, several of the acute transforming viruses (with the exception of Rous sarcoma virus) are replication-incompetent, whereas leukemia viruses replicate readily in vitro and in vivo.
The advent of recombinant DNA techniques in late 1970s allowed the molecular cloning and sequence analysis of the two types of viruses, which provided a molecular explanation of their biological differences. These studies showed that the leukemia viruses encode three polypeptides, named gag, pol and env. Of these, the gag gene encoded a polypeptide chain that is cleaved proteolytically into smaller polypeptides that from the core proteins of the virion.
The pol gene encodes a polypeptide chain that is cleaved into two polypeptides, one of which is the reverse transcriptase that enables the virus to convert its RNA into DNA. The second polypeptide fractions as an integrase that allows the pro viral DNA to integrate into the host viral genome. The env gene encodes the viral envelope protein that plays an important role in virus- host cell membrane interactions.
In addition to these three genes, the retroviral genome contains a stretch of sequences that are duplicated at the 5′ and 3′ ends of the provirus, termed Long Terminal Repeats (LTRs). The LTRs contain sequences that constitute some of the most potent eukaryotic promoter/enhancer elements in mediating high-level transcription of viral RNA.
Comparison of the structures of the acute transformation and leukemia viral genomes revealed that the acute transforming viruses contain an additional segment of genomic RNA not present in leukemia viruses. Deletion of this sequence leads to loss of the transforming ability of these viruses. These observations suggested that a protein encoded by this unique piece of genetic material is responsible for inducing of tumors in animals.
The first of these genes was originally found in the Rous sarcoma virus and was designated as the src oncogene. Structural analysis of several of the acute transforming viruses revealed that acute transforming viruses very often suffer deletions in their env, pol, and/or gag sequences, which explains their inability to replicate in vitro or in vivo.
The only exception to this rule is the Rous sarcoma virus, which contains all three structural genes of the avian leucosis virus, in addition to the Ere gene, and thus constitutes the only replication-competent acute transforming virus.
An important breakthrough in cancer research came from the discovery that oncogenes of acute transforming viruses are in fact derived from normal cellular DNA and that this genetic information is transduced by acute transforming viruses via genetic recombination. An analysis of normal cellular DNA by Stehlin and his coworkers (2), using a probe derived from the src oncogene (derived from Rous sarcoma virus), revealed the presence of endogenous oncogene-related sequences in normal chicken DNA Following the lead provided by the Rous sarcoma virus, retro-virologists have isolated more than 200 different tumor-producing, acute transforming viruses from animal tumors of different species.
Table lists some of the acute transforming viruses and the oncogenes transduced by them. An examination of this table shows that several of the acute transforming viruses isolated from different animal species contain the same oncogene, suggesting that there are only a finite number of transforming genes in the avian and mammalian genomes and that these often undergo recombination with replicating retroviruses, leading to the formation of acute transforming viruses.
Since the first identification of the v-src oncogne, a number of different approaches have been used to identify genes responsible for the altered growth properties of tumor cells. Now, the term “oncogene” is used more broadly to include any gene whose expression is associated with enhanced growth of tumor cells.
8. Carcinogenic Risk Assessment:
Assessment of carcinogenic risk involves a number of steps- (1) hazard identification; (2) mechanism elucidation; and (3) quantification of risk based upon the mechanism.
It as has been suggested, all substances are hazardous, then the methods used for what we refer to as ‘hazard identification’ are, in fact, telling us something about the potency of the chemical. With all its difficulties, the ‘hazard identification’ process remains the easiest part of the process leading to quantitative risk assessment.
Mechanism of action is the next, extremely important undertaking and must involve a broad range of disciplines, including pathologists, toxicologists, metabolic chemists, pharmacokinetics specialists and molecular biologists. Mechanistic studies have always been interesting, but they have now achieved a very high level of importance in the evaluation of the toxicology of a compound.
Today it would be difficult to overestimate their importance. There are few general principles which can be applied to understanding mechanisms of action, each carcinogenic entity being studied on a case-by-case basis, although genetic toxicity has been a high-profile consideration in most mechanistic studies.
Nevertheless, each chemical structure is unique and it is a serious error to attempt to group chemicals without experimental evidence – on the basis of either their structural similarities or similarities in their effects. Chemicals sharing particular active groups can have very different effects and particular effects may be achieved by different mechanisms.
Quantification of risk is the ultimate step, although investigators in this field do not pretend that a solution to the problem is at hand. The best that they can offer at the moment is mathematical models which fit the observed data and may have a foundation in biology.
Active efforts need to be constantly directed towards the reduction of environmental pollution by carcinogens and other immunosuppressive toxicants. This will reduce the chances of human cancer by environmental carcinogens. Judicious adoption and control of changed life style (smoking, drinking, using sophisticated foodstuff with preservatives etc.) is also needed to prevent environmental pollution. Environmental pollution is caused by human beings which is preventable. Therefore, environmental linked cancer too (90% of human cancer) can be kept well under control.
The use of anticarcinogens and host immuno-stimulation are the promising areas and should be clinically tried to prevent carcinogenesis.