Mechanisms of Environmental Carcinogenesis

Mechanisms of Environmental Carcinogenesis P Irigaray, Association for Research and Treatments Against Cancer, Paris, France D Belpomme, Association for Research and Treatments Against Cancer, Paris, France and Paris-Descartes University and European Hospital Georges Pompidou, Paris, France & 2011 Elsevier B.V. All rights reserved.

Abbreviations AhR BBP CMR CYP DEHP EBV EMF HBV HCV HHMMTV HHV HIV HPV HTLV-1 IARC NAT NOC PAH PCB ROS UV

aryl hydrocarbon receptor benzyl butyl phthalate carcinogenic, mutagenic, and reprotoxic cytochrome P450 diethylhexyl phthalate Epstein–Barr virus electromagnetic field hepatitis B virus hepatitis C virus human homologue of mouse mammary tumor virus human herpes virus human immunodeficiency virus human papilloma virus human T-cell lymphotropic virus type 1 International Agency for Research on Cancer N-acetyltransferase N-nitroso compound polycyclic aromatic hydrocarbon polychlorobiphenyl reactive oxygen species ultraviolet

Introduction The increasing incidence of a variety of cancers after the Second World War (WWII) confronts scientists with the question of their origin. It has been previously shown that demographic expansion and aging as well as progress in cancer detection using new diagnosis methods and screening tests cannot fully account for the observed growing incidence. Moreover, many environmental factors have been listed rated as certainly or potentially carcinogenic by the International Agency for Research on Cancer (IARC) that, in addition to lifestyle-related factors, may in fact be involved in human carcinogenesis. Therefore, the hypothesis has been put forth that the post-WWII change in environment may have contributed to the increased number of cancer detected and consequently that environmental carcinogens – that is, viruses and other microorganisms, radiation, and xenochemicals – may play a more important role in carcinogenesis that it was expected.

In this article, the different mechanisms have been described whereby environmental carcinogens may induce and generate cancers. This article first reviews the present theories of cancer and analyzes the general mechanisms of carcinogenesis. Then analyzes the specific and common mechanisms whereby viruses, radiation, and environmental chemicals can contribute to cancer.

What are Environmental Carcinogens? Environmental carcinogens are defined as physical, chemical, and biological exogenous agents that cause cancer after having penetrated into the organism through several possible routes: respiratory (air pollutants), digestive (food contaminants and additives), cutaneous (radiation and cosmetics), sexual (viruses), and other (including fetal contamination by maternal blood during pregnancy). The risk fraction attributable to environmental carcinogens is still a matter of controversy. There are currently two opposite interpretations of the growing incidence of cancer. The classical interpretation considers that environmental carcinogens can only make a minor contribution to overall cancer incidence changes and therefore that increase in cancer detection and life expectancy as well as lifestyle-related influences can explain the current growing incidence of cancer. Conversely, challenging interpretation is that the contribution of involuntary exposure to multiple and diverse environmental carcinogens accounts for a significant portion of the increase. Such a new theory mainly results from the observation that environment has changed over the same timescale as the recent rise in cancer incidence and that this change caused the accumulation of many new carcinogenic and cocarcinogenic agents in the environment.

Distinction between Exogenous and Endogenous Carcinogens As they come from the environment, exogenous carcinogens must be distinguished from endogenous carcinogens, which by definition result from the normal metabolism of individuals who are not exposed to a polluted environment (i.e., that inhale nonpolluted air, ingest noncontaminated food, are not submitted to radiation and are

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not contaminated by pathological microorganisms). Endogenous mutagens mainly include free oxyradicals and particularly aldehydes and ketones that naturally arise from respiration, or that may be contained in food or that are molecular intermediates issued from the metabolism of food or from the metabolism of bacteria of the endogenous microflora. A linkage point, however, is that oxidative DNA damage caused by these natural potentially carcinogenic endogenous molecules can be easily repaired, whereas DNA alterations resulting from exogenous carcinogens are generally not correctly repaired by the different repair systems and so are mutagenic and carcinogenic (see in the following text). The distinction between exogenous and endogenous carcinogens may therefore be critical for the determination of the cause of cancers, since endogenous carcinogens might be associated with a limited number of sporadic cancers occurring spontaneously whereas exogenous carcinogens, including oncogenic viruses, radiation, and xenochemicals, may cause numerous acquired sporadic cancers. Thus, Knudson’s classification was modified and three main categories of cancer according to their causal origin were distinguished: hereditary cancers resulting from high penetrant inherited germinal mutations, which represent no more than 1% of overall cancer cases (Group 1); spontaneously occurring sporadic cancers resulting from a possible mutagenic effect of naturally occurring endogenous carcinogens or from spontaneous mutations (Group 2) (see in the following text); and sporadic cancers caused by exogenous carcinogens, which include tobacco smoking-related cancers and environmental cancers, which may represent the majority of cancer cases overall (Group 3) (Table 1).

Distinction between Lifestyle-Related Risk Factors and Cancer-Causing Agents To better delineate the respective contribution of endogenous and exogenous factors in the process of carcinogenesis, lifestyle-related factors should be clearly distinguished from cancer-causing agents. Several lifestyle-related behaviors including sun exposure

Table 1

Group I Group II Group III a

(ultraviolet, UV), multiple sexual partnerships (risk of sexual transmission of papilloma and hepatitis B viruses (HBVs)), and tobacco smoking (chemical mutagens and promoters in smoke and tars) are examples of lifestyle-related risk factors involving genuine exogenous carcinogens. To clarify the role of the different factors involved in the process of carcinogenesis, it has been proposed to distinguish lifestyle-related risk factors, as determined by epidemiological studies, from cancercausing agents, as mainly determined by toxicological and biological studies. Indeed, lifestyle-related factors are not cancer-causing agents, but risk behaviors that may contribute to the direct or indirect action of cancercausing agents (e.g., exogenous carcinogens in smoke and tars resulting from the combustion of tobacco, radiation, and viruses) and therefore to cancer occurrence.

General Mechanisms of Carcinogens Spontaneous Carcinogenesis Theoretically, in any dividing cells, spontaneous mutations is supposed to arise from miss-copying of damaged DNA template or from inaccuracy of DNA replication, when there is no pre- and postreplication faithful DNA reparation. However, these stochastic events – wear-associated errors occurring in DNA synthesis and replication – are normally very infrequent and easily repaired, thus too low to account for the high incidence of cancers; as commonly observed in humans and animals. In addition, as previously indicated, endogenous carcinogens may contribute to the spontaneous occurrence of some sporadic cancer. Indeed, on the basis of analysis of old medical literature and observations of animal breedings, it is assumed that most cancers do not occur spontaneously but are caused by acquired factors, among which exogenous carcinogens may act predominantly. Distinction between Genotoxic and Nongenotoxic Carcinogens Cancer is generally defined as a multistep process involving the accumulation of mutations in specific genes that lead an initial clone of transformed cells to

Proposed revised Knudson’s classification of cancer according to genetic and environmental factors Etiological viewpoint

Endogenous carcinogens

Exogenous carcinogensa

Genetic factors

Hypothetical attributable fraction

Hereditary cancers Spontaneous cancer occurrence Acquired carcinogenesis

? þþþ

? 0

Germinal mutations ?

B1% B20–10%

?

þþþ

Genetic polymorphism

B80–90%

Include tobacco smoking-associated carcinogens and environmental carcinogens.

Mechanisms of Environmental Carcinogenesis

irreversibly progress and expand in the organism. DNA mutations are indeed a critical rate-limiting step in carcinogenesis. Based on the sequencing analysis of the human cancer genome, recent data identifying genetic alterations in cancer cells have considerably reinforced the somatic mutation theory of carcinogenesis. At a molecular level, mutations of the three major candidate cancer genes – oncogenes, tumor suppressor genes, and DNA mismatch repair genes – have been described. Yet, it has been proposed that aneuploidy – the occurrence of DNA or chromosome rearrangements during mitosis – plays a more critical role in carcinogenesis than point mutations or small mutations and that only ‘driver’ mutations – that is, mutations that confer a clonal cell growth advantage – are determinant for cancer progression. Moreover, it has been recognized that all DNA lesions do not necessarily induce mutations and that a prerequisite for mutagenesis is that cells with DNA lesions absolutely need to survive and divide. Furthermore, silencing of gene expression due to temporary cell dysfunction or to epigenetic changes, such as aberrant hyper or hypomethylation of DNA, may also indirectly cause mutations. Indeed, it has been proposed that carcinogenesis is more than mutagenesis, that is, in addition to mutagenesis, many epigenetic alterations contribute to carcinogenesis. These considerations led to attempt at characterizing carcinogens, especially environmental carcinogens, according to their genotoxic or nongenotoxic potentials. A genotoxic carcinogen is a carcinogen that directly causes stable DNA damage, which cannot be faithfully repaired and which therefore results in mutation after cell division; whereas a nongenotoxic carcinogen does not interact with DNA, but can affect gene expression, cell functions, and modify the normal phenotype through epigenetic alterations. Nongenotoxic carcinogens are frequently characterized by pleiotropic effects. They can promote cell division and cell survival or contribute to cell transformation or tumor progression. Moreover, some nongenotoxic carcinogens may in fact be indirectly mutagenic, by inducing secondary mutations. Cells are indeed extremely vulnerable to gene dysfunction during mitosis and this is particularly the case during fetal development where tissue alteration and disorganization can occur following exposure to xenochemicals. Consequently any agent that acts through epigenetic mechanisms, albeit it does not interact with DNA may indirectly induce mutations and particularly aneuploidy. However, genotoxic carcinogens may also be associated with promoting effects, so that repeated exposure to such agents may be an effective way to induce cancers in experimental animal models. Therefore, it was concluded that the distinction between genotoxic and nongenotoxic carcinogen is questionable and generally insufficient to account for a precise definition of the mechanisms of action of carcinogens.

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Distinction between Carcinogens and Cocarcinogens; and among Carcinogens, between Tumor Initiators, Promoters, and Progressors By definition, carcinogens are not cocarcinogens. A carcinogen is defined as a genotoxic or nongenotoxic cancer-causing agent; whereas a cocarcinogen is not carcinogenic itself, but it can activate a carcinogen or enhance its carcinogenic effects. Accordingly, oncogenic viruses, radiation, and xenochemicals such as benzo[a]pyrene, aromatic amines (AAs), and heterocyclic amines (HAAs), N-nitroso compounds (NOCs) and dioxins such as the prototypical 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) are environmental carcinogens; whereas exogenous agents that deplete the organism of endogenous detoxifying molecules such a glutathione (GSH) or that activate environmental procarcinogens into carcinogens through the induction of cytochrome P450 (CYP) enzymes are cocarcinogens. However, carcinogenesis is an extremely complex multifactorial, multigenic, and multistage process. On the basis of experimental data, this process has been modeled into three sequential and successive phases  initiation, promotion, and progression  during which promotion is a classically reversible rate-limiting phase, because it determines the latent period of premalignant tumor formation. Accordingly, carcinogens have also been distinguished into tumor initiators, promoters, and progressors. Tumor initiators are defined as carcinogens capable to induce a first irreversible driver mutation in a single stem cell or progenitor cell, through direct or indirect mutagenesis, so an initial clone of initiated cells can emerge (see in the earlier text); tumor promoters, as nongenotoxic agents capable to cause clonal expansion of initiated cells, that is, to induce a reversible proliferation of mutated cells and to prevent these cells from apoptotic loss, so the possibility of additional genetic and epigenetic changes is preserved; and tumor progressors as carcinogens that irreversibly contribute to the acquisition of the necessary complete phenotype hallmarks of transformed cells, that is, the capacity of these cells to invade normal tissues, to induce neoangiogenesis, to organize themselves as a tumor in association with host-related stroma cells, and finally to progress and metastasize in the organism. Consequently, mutagens, which comprise initiators and progressors, can theoretically be clearly distinguished from promoters; whereas among carcinogens, promoters may be difficult to distinguish from cocarcinogens. Moreover, due to their cellular pleiotropic effects and multiple mechanisms of disturbance of tissue homeostasis, tumor promoters may also be secondarily genotoxic and thus difficult to distinguish from mutagens. A typical example of exogenous chemical promoter is the phorbol ester, phorbol 12-myristate 13-acetate (TPA), which has

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been characterized by numerous phenotypic biological properties. Also, environmental promoters such as endocrine disruptors or immunosuppressors may be associated with pleiotropic effects. Many endogenous hormones or growth factors have also been shown to be tumor promoters, so it clearly appears that tumor promoters may in fact be from endogenous or exogenous origin. Because mutations can not occur in nondividing cells, endogenous and exogenous tumor promoters are absolutely necessary as long as the clone of premalignant cells is not becoming promoterindependent. Therefore, during initiation and promotion mutagens and promoters intimately cooperate, so that a critical number of driver mutations can be reached; leading the premalignant clone of transformed cells to become promoter-independent, thus fully malignant. Finally, although some exogenous carcinogens have the capacity to induce and generate all three phases of the carcinogenesis process – they are called complete carcinogens – many others are ‘partial’ carcinogens because they need to act together to generate the complete process. An example is cigarette smoke and tars that contain mixtures of different mutagenic and tumor promoting cancer-causing agents equivalent to a complete carcinogen, so tobacco smoking is a lifestyle-related risk factor that fully contributes to induce and generate cancers. A similar observation may apply to the numerous exogenous chemicals present in the environment, where they exist as mixtures of all types of carcinogens and cocarcinogens, so they may interact with each other (cocktail effects). Table 2 summarizes the general mechanisms of environmental factors that are involved in carcinogenesis according to their mutagenic, promoting or cocarcinogenic effects.

Specific Mechanisms of Environmental Carcinogens Viruses and other microorganisms, radiation and many xenochemicals can cause cancer through diverse and specific mechanisms. BBP, benzyl butyl phthalate; DEHP, diethylhexyl phthalate; HHMMTV, human homologue of mouse mammary tumor virus; HHV, human herpes virus. Viruses and Other Microorganisms Viruses can induce and generate cancer through two distinct mechanisms, that is, directly by inducing mutations and indirectly by inducing inflammation or immunosuppression. Oncogenic DNA viruses can induce mutations by inserting their own genomic DNA into the cell DNA, whereas oncogenic RNA viruses induce mutations by inserting a complementary DNA copy of their RNA genome into the target cell DNA, thanks to the

Table 2 Proposed classification of exogenous environmental agents according to their carcinogenic and cocarcinogenic properties

Microorganisms EBV HBV/HCV HHMMTV HHV-8 HIV HTLV-1 HPV Helicobacter pylori Radiation Radioactivity UV EMF Particles and xenochemicals Air fine particlesa Asbestos Arylaminesb Azoı¨c dyes Bisphenol A b-Naphylamine Benzene and derived molecules DEHP, BBP Dioxins Formaldehyde and derived Hormonal residues Metals, metalloids N-nitroso compoundsc NO2 Organochlorines PAHsd PCB Pesticidese Vinyl chlorides (monomers)

Mutagen

Promoter Cocarcinogen

M M M

P

P

C C

P

C

P P P

C C C

M M

M M M

M M M M M M M M M

C C

P C P P

C

P M M M M (X5 rings) M (some) M (some) M

C P P P (o5 rings) P P P

C C

a

Air carbonaceous particles, especially PMo2.5, are vectors for chemicals, including PAH and organochlorines (pesticides). b Include aromatic amines (AAs) and heterocyclic aromatic amines (HAAs). c Nitrates, nitrites, nitrosamines, nitrosamides. d PAHs of high molecular weight (five to seven rings) induce DNA adduction processes and so are mutagenic, whereas PAHs with low molecular weight (two to four rings) are nongenotoxic promoters. e Act usually as endocrine disruptors or immunosuppressors (promoters), but some of them can be also mutagenic. BBP, benzyl butyl phthalate; DEHP, diethylhexyl phthalate; HHMMTV, human homologue of mouse mammary tumor virus; HHV, human herpes virus.

presence of a RNA-dependent DNA transcriptase reverse. There are three groups of double-stranded DNA viruses, that is, the human papilloma viruses (HPV) – mostly HPV type 16 – the hepatitis-B virus (HBV), and the Epstein–Barr virus (EBV). Furthermore, there are two groups of diploid RNA viruses, that is, the Hepatitis C

Mechanisms of Environmental Carcinogenesis

Virus (HCV) and the human T-cell lymphotropic virus type 1 (HTLV-1), which have been shown to be associated with several human solid cancer and leukemia. There are several different mechanisms whereby oncogenic viruses can induce mutations. A direct mechanism is the insertion of one or several viral oncogenes into the cell DNA or after insertion, the activation of cellular protooncogenes into oncogenes. Other mechanisms are possible: while HPV-16 has been shown to be mutagenic by inserting the viral oncogenes E6 and E7 into cell DNA, thus producing proteins that inhibit p53; HBV is thought to be mutagenic by producing reactive oxygen species (ROS). Likewise, although the retrovirus HTLV-1 has been shown to be directly mutagenic, HCV as HBV is thought to be indirectly mutagenic by producing ROS in infected cells. Oncogenic viruses are defined as viruses that directly induce mutations. However, nononcogenic viruses may also play an indirect role in carcinogenesis, through the induction of immunosuppression or inflammation. This has been clearly demonstrated for the two human immunodeficiency viruses (HIVs), which, albeit they are not mutagenic, have been classified as carcinogenic by IARC, because they promote the mutagenic effect of oncogenic viruses through immunosuppression induction. Also, infectious but nonmutagenic viruses might be involved in the carcinogenic process of some presumably virus-induced neoplasia, such as the common form of childhood acute leukemia. In addition, some microflora bacteria of the gastrointestinal tract – such as Helicobacter pylori for gastric cancer, parasites such as Opsthorchis viverrini for gallbladder cancer, and Shistosoma haematobium for bladder cancer – have been shown to be cofactors causally implicated in carcinogenesis, through inflammation induction and free radicals production. Finally, an important finding is that several types of microorganisms can intimately cooperate in carcinogenesis. For example, it has been shown that in EBV endemic infection area, environmental tumor promoters such as extracts of a commonly used plant, Euphorbia tirrucali, and mosquitoes-dependent infections such as malaria and arbovirus infection are cofactors that cooperate for the genesis of Burkitt lymphoma. Likewise, it has been shown that HBV infection and aflatoxin exposure cooperate for the genesis of hepatocellular carcinoma. Aflatoxins are produced by the contaminated moulds Aspergillus flavus and Aspergillus parasiticus. Transgenic mouse models that contain HBV targeted to the liver have evidenced that the frequency of hepatocellular carcinoma increases with aflatoxin exposure. Moreover, it was found that the relative risk for developing hepatocellular carcinoma was 3.4 for people exposed to aflatoxins, but it was 59.4 for people that in addition to aflatoxin exposure had tested positive for hepatitis B.

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It is estimated that oncogenic viruses are involved in approximately 16% of human cancers worldwide, whereas it would only range 5% in high-income countries. On the basis of previous analysis, it can be assumed that virus-induced cancers in humans are probably more frequent in high-income countries than it is usually recognized. In these countries, they might range up to 10–15% of total cancer. Ionizing and Nonionizing Radiation Radiation can cause cancer by inducing mutations and also contribute to carcinogenesis by causing promotion or cocarcinogenic effects, through immunosuppression induction. Radiation-induced cancers are stochastic late effects of ionizing and nonionizing radiation. They include some leukemias and lymphomas, thyroid cancers, skin cancers, some sarcomas, some lung and breast carcinomas, and some brain tumors. Studies of radiation-exposed populations were initially based on occupational exposure. This began to change with the study of the survivors of atomic bombs exploded above Hiroshima and Nagasaki. Moreover, improved understanding regarding the molecular basis of radon-induced cancers has provided support for considering low radon level as a cause of approximately 10% of lung cancers. Ionizing radiation induces point mutations, dimerization, and major chromosomal changes involving DNA breakages and rearrangements. Exposure to low linear energy transfer radiation increases the frequency of chromosome aberrations proportional to the square of the radiation dose. However, radiation-induced cancers depend on many variables, and – as for low-dose chemicals – low dose of ionizing radiation must be considered significant risk of somatic heritable mutations. Nonionizing radiation comprises UV rays and pulsed electromagnetic fields (EMF). Exposure to UV is a dosedependent risk factor that can cause skin cancers and melanoma. UVB can directly cause mutations, whereas UVA can indirectly damage DNA through ROS production. Pulsed EMF of very low frequency or extremely low frequency and of radiofrequency have been the object of many scientific debates to answer the question whether they can induce cancers. Despite differences in study design and setting of epidemiological studies, recent results appear to be sufficiently convincing to consider that children living near high-voltage power lines are at a relative increased risk of leukemia; and that daily prolonged use of mobile phones during a X10-year long-term period is associated with increased risk of brain tumors. Because direct mutagenesis depends on energy and energy level is not sufficient to cause direct breakage of DNA, an alternative explanation is proposed, that pulsed EMF may be indirectly mutagenic by

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inducing epigenetic changes. As discussed in a recent international consensus meeting, EMF-related health end points include several types of biological responses such as genotoxicity, immune system deregulation and inflammation, and several types of related diseases such as cancers, particularly childhood leukemia, brain tumors, and breast carcinoma. Because EMF-related biological responses are extremely complex, it has been suggested that, according to the second principle of thermodynamics, they may in fact depend on entropy – that is, loss of structural information – rather than energy-related effects, consequently causing epigenetic dysregulation and structural tissue disorganization. Here too, a possible indirect molecular mechanism for epigenetic disregulation, tumor disorganization, and carcinogenesis could be the production of free radicals. This might explain why EMF-related cancers seem to require a longer period of exposure than ionizing radiation and why EMF-related cancers appear overall to be associated with a weaker relative risk. Overall, it may be hypothesized that radiation-related cancers might represent up to 10% of total cancer cases. There is, however, no clear assessment of this populationattributable risk, which needs to be further investigated. If it is assumed that approximately 25% of cancer cases overall are associated with tobacco smoking, 15% are virus-induced, and 10% are radiation-induced, approximately 50% of the total number of cancer cases might be caused by chemicals and the fraction of cancer attributable to the environment might be in the order of 75% of overall cancer cases. Xenochemicals The petrochemical age and other dangerous aspects of the industrial revolution, in full gear by the second half of the twentieth century, had consequences in domains such as energy, transport, agriculture, food, and health; this caused the synthesis, production, and introduction into the environment of millions of tons of tens of thousands of different man-made xenobiotic chemicals. Such products can contaminate air, soil, water, and food and be persistent pollutants in the environment. Many pollutants are carcinogenic, mutagenic, and teratogenic substances, which therefore act as mutagens, tumor promoters or both, or as cocarcinogens. They can play a major role in the genesis of many cancers and thus may account for their currently growing incidence. A wide variety of xenochemicals and chemical classes can cause cancer in animals and humans. Experimental animal models have reproduced every major type of human cancer, showing that exposure to specific chemical carcinogens can induce organ-specific tumors.

Lipophilicity as a basic property of many organic pollutants

Chemical-related DNA damage can occur directly from environmental exposure, or indirectly after metabolic activation of xenochemicals to DNA-reactive molecules. A basic condition for activation and DNA damage is that exogenous chemical carcinogens enter cells. All nonplant organisms use their cell membrane as a hydrophobic permeability barrier to control access to their internal milieu. Diffusion across cell membrane of polar (hydrophilic) pollutants is mediated through transport proteins that specifically select substrates from the extracellular milieu, so polar compounds can not enter cells if they are not recognized by specific transporters. If they are not metabolized into polar molecules by specific detoxification enzymes, nonpolar (hydrophobic) pollutants can enter cells. Because the organism is frequently not able to fully metabolize man-made nonpolar pollutants, many of them, such as polycyclic aromatic hydrocarbons (PAHs), dioxins and polychlorobiphenyls (PCBs), can enter cells, due to their lipophilic properties; consequently they can bioaccumulate in the adipose tissue of multicellular organisms and thus may contaminate many trophic ecosystems including the whole human food chain. DNA adduction and mutagenicity

A major specific and basic property of exogenous chemical carcinogens is that they can induce stable and irreversible bulky adducts – that is, covalent bonds with macromolecules – many of which DNA adducts they form can not be correctly repaired by the cell repair systems; whereas as previously indicated endogenous chemical carcinogens form adducts (particularly DNA adducts) that can be normally easily repaired. An explanation could be that most exogenous carcinogens or their metabolites are ‘hard’ electrophiles that may irreversibly adduct ‘hard’ nucleophilic sites on DNA, whereas endogenous molecules such as unsaturated aldehydes and ketones are ‘soft’ electrophiles that reversibly react with ‘soft’ nucleophiles on the DNA. Because there is a good correlation between the ability to form stable DNA adducts and the capacity to induce tumors in animals, DNA is considered as the ultimate target for most chemical carcinogens. Mutagenic carcinogens may transfer simple alkyl or (complexed) arylalkyl groups to specific sites on DNA bases, or transfer arylamine residues to DNA. Metabolic activation to yield DNAreactive alkylating and arylalkylating agents involves oxidation at carbon atoms, whereas to yield DNAreactive arylaminating agents it involves either oxidation or reduction at nitrogen atoms. Among alkylating and arylalkylating agents are PAHs, NOCs, and aliphatic epoxides, whereas among arylaminating agents are AAs and HAAs and aminoazodyes. DNA-reactive chemicals

Mechanisms of Environmental Carcinogenesis

are usually mutagenic, but as aforementioned carcinogenicity is more than mutagenicity. In a serial analysis of chemicals, 16% of tested carcinogens were not found to be mutagenic, whereas 66% of noncarcinogens were found to be mutagenic. Furthermore, because the interaction of genotoxic carcinogens with DNA has been thought not to be random, it has been hypothesized that mutagenic xenochemicals may induce some specific and reproducible mutations. In fact, this ‘fingerprint’ hypothesis has not been validated, because most mutagenic xenochemicals actually can form several types of mutations depending on the conformation of DNA, the type, and the location of the adducts. Metabolic activation of environmental chemical carcinogens

A number of metabolic pathways activate or detoxify exogenous chemical carcinogens. These pathways are complex and interactive. Many enzymes involved in these pathways are inducible and their activity may be thus modified by additional environmental exposures, hormones and diet, adding a further complexity to the process of chemical carcinogenesis. Normally, the host is able to detoxify many chemical environmental pollutants thanks to phase I, II, and III enzymes, and other proteins such as GSH and ATP-binding cassette (ABC) efflux proteins; all being involved in the metabolism of xenobiotics. However, during this process, procarcinogens can be transformed into active carcinogens – more precisely into promoters or mutagens – by several enzymes. A basic general mechanism of biotransformation has been put forward pointing out that a parent molecule – generally a ‘soft’ electrophile – may be converted into an oxidative metabolite, which is a ‘hard’ electrophile, so that the parent molecule and its oxidative metabolite exhibit distinct electrophilic capacity. This difference in electrophilicity therefore accounts for the different nucleophilic target of the parent molecule and of its metabolite, and can predict whether any molecule can be an adduct to DNA. Among phase I detoxifying enzymes that mainly contribute to the activation of chemical carcinogens is the CYP system that acts in addition to phase II conjugating enzymes such as N-acetyl transferases (NATs) and sulfotransferase, which are important carcinogen-activating ubiquitous intracellular enzymes. The CYP system, which comprises more than 40 isoforms, can activate high molecular weight PAHs (more than 4 rings; h PAHs), nitrosamines and other NOCs, and AAs and HAAs, whereas peroxidases (phase I enzymes) can activate AAs. In addition, a phenotype of slow or fast metabolic activation may lead to different cancers. A genotypically recessive slow acetylation phenotype involving NAT1 has been found to be associated with occupationally

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induced bladder cancer in dye workers exposed to AAs, whereas a genotypically dominant rapid acetylator phenotype, involving both NAT1 and NAT2 has been found to be associated with colon cancer in people exposed to dietary HAAs. Moreover, in addition to these different substrates, interethnic and interindividual genetic polymorphisms are major contributing factors that determine the type and quantity of synthesized enzymes and therefore the type of pathways and the intensity of the activation process. For many carcinogens, the activation process takes place in the host. This is the case for benzo[a]pyrene and other h PAHs for which the activation of CYP1A1 or CYP2D6 or CYP2E1 has been found to be associated with an increased risk of lung cancer. Endogenous bacteria may also contribute to activate carcinogens. This is, for example, the case for AAs, for which the CYP1A2-induced hydroxylamine is deconjugated in the colon by a bacterial glucuronidase, so hydroxylamine can be acetylated by NAT2. This is also the case for NOCs. Nitrates are not per se carcinogenic. However, nitrates can be transformed into nitrites through nitrosation by the microflora bacteria of the digestive tract, then nitrites can be transformed into the highly mutagenic NOCs, alkylnitrosamines and alkylnitrosamides, which are further activated by CYP2E1, CYP2A6, and CYP2D6 to form stable DNA adducts in target tissue. The central role of the AhR-activating and -inducible CYP systems in environmental chemical carcinogenesis

A number of xenochemicals that cause cancer in laboratory animals are not demonstrably mutagenic. These xenochemicals are environmental pollutants such as dioxin (prototypically 2,3,7,8-TCDD), dioxin-like PCBs, organochlorine pesticides, and low molecular weight PAHs (2, 3, or 4 rings; l PAHs) that can act as promoters or cocarcinogens. A basic finding is that many xenochemicals such as PAHs, as well as dioxins, dioxin-like PCBs, and other organochlorines act through a common ubiquitous molecular pathway involving the arylhydrocarbon receptor (AhR). AhR is a ligand-activated transcription factor known to mediate the pleiotropic effects of many environmental pollutants. Pollutants that combine with and activate AhR cause the transcription of many genes involved in cell proliferation, cell differentiation and cell survival, and consequently induce a broad spectrum of systemic promoting effects. In addition, a major event following AhR activation is the activation of several CYP response genes, causing cocarcinogenic effects. Because the CYP system is a major determinant for the activation of many environmental chemical carcinogens, both the AhR-activating and the inducible CYP systems are central in environmental chemical carcinogenesis. In addition to inducing promoting and

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Mechanisms of Environmental Carcinogenesis

cocarcinogenic effects, some environmental chemical carcinogens may also induce mutagenic effects. Metals and metalloids as environmental carcinogens

Several metals and metalloids have been rated as certain or probable carcinogens by IARC, albeit their mechanisms of action are not clear. Metals and metalloids could act as cocarcinogens by activating procarcinogens in the liver or by increasing the promoting effect of estrogens. They could also act by replacing the natural enzyme-complexed metal, thus inactivating the metabolic pathway and function of key enzymes. Carcinogenic metals and metalloids, such as arsenic, cadmium and nickel, and some putative carcinogens such as cobalt and lead can inhibit zinc finger-containing DNA repair proteins. Damage of zinc finger in DNA repair proteins can be regarded as a novel mechanism in carcinogenesis. Moreover, some metals and metalloids may also be mutagenic through other mechanisms. Indeed many of them can interact with DNA. Metal compounds such as chromium(VI) are taken up by cells as chromate anions and are reduced intracellularly via reactive intermediates to stable Cr(III), which can directly interact with DNA. These Cr(III) intermediates may affect DNA by terminating replication or reducing replication fidelity, thus leading to mutations. Cr(III) can also form DNA-proteins and DNA-amino acids and glutathione cross-links. Platinum compounds (i.e., cis-diaminedichloroplatinum) are well known DNA strand breakers. They can form DNA cross-links and DNA-protein cross-links leading to mutations. There is evidence that nickel may act via an epigenetic mechanism involving heterochromatic regions of the genome. Finally, many studies have focused on metal-induced carcinogenicity, emphasizing the mutagenic role of metals such as iron, copper, chromium, nickel, cadmium, and arsenic in carcinogenesis, through the production of ROS. Metal-mediated formation of free radicals can indeed cause various modifications of DNA bases and other intracellular molecular changes that can contribute to carcinogenesis. A typical example is asbestosis-induced cancers that may be caused by the generation of free radicals due to the presence of oxidative iron.

Search for Basic Properties and Mechanisms Common to Environmental Carcinogens Basic assumptions of environmental carcinogens are that they act at repeated low doses, and that chronic exposure is more relevant than dose intensity. Furthermore, environmental carcinogens may act according to several common cellular and molecular mechanisms.

Low-Dose Effects and Role of Exposure Duration It is commonly claimed that environmental carcinogens, especially radiation and xenochemicals, are not in sufficient quantities in the environment to reach exposure levels that can cause cancer. However, there are many counter arguments to this claim. Cancer is indeed a disease basically caused by chronic exposure to low-dose carcinogens. As for radiation, there is no safe dose threshold, for mutagenic xenochemicals, meaning that they can induce mutations at extremely low levels. Yet, a similar consideration is evidenced for environmental organochlorine pollutants such as dioxins and for metals and metalloids and more generally may apply to tumor promoters and environmental endocrine chemical disruptors, for which the promoting effect depends on the sensitivity of receptors. Indeed, for these molecules there is a nonmonotonic inverted U or J-shaped dose-response relationship, indicating more risk at low than at high dose levels. Similar consideration might apply to viruses for which a few number of particles with infectious capacities may result in malignant transformation or immunosuppressive effects. Consequently, it appears that environmental carcinogens, be they mutagens or promoters, would in fact be carcinogenic at doses lower than those at which no effect level is observed in classical rodent tests. In addition, since many environmental organic pollutants can bioaccumulate in the adipose tissue (see in the following text), it has been proposed that they can be released in the blood circulation at doses which do not correspond to those found in the environment and thus may be carcinogenic at environmental extremely low doses. Moreover, according to the current concept of carcinogenesis duration of exposure (i.e., repeated low doses) rather than dose intensity of carcinogens should be considered. Indeed, it clearly appears that the older a person is, the longer will be his/her exposure period to carcinogens and hence the greater the probability that s/he will get cancer. Chronic Inflammation and Immunosuppression as Cellular Mechanisms Common to Many Environmental Cancer Types Environmental carcinogens can cause cancer through the induction of several pathological conditions such as inflammation and immunosuppression. Nononcogenic as well as oncogenic viruses, other microorganisms, radiation, and xenochemicals can induce cancer through the induction of chronic inflammation, more precisely through the production of ROS and other free radicals such as nitric oxide and hypochlorite by phagocytes and neutrophils and through a cascade activation of many pro-oxidant cytokines and growth factors. It is only recently that gastritis, ulcerative colitis, chronic

Mechanisms of Environmental Carcinogenesis

pancreatitis, and hepatitis have been recognized as risk factors that may contribute to the genesis of many cancers, may be about one-third of all cancers. Environmental carcinogens that elicit an inflammatory response are potent generators of humoral immunity that cooperate with cellular immunity and effectively suppress antitumor immune response, while simultaneously enhancing angiogenesis. In addition, generation of ROS can damage host cells and DNA, and can be associated with promoting effects. Consequently, environmental agents that induce inflammation could contribute to tumor initiation, tumor promotion, and tumor progression. In addition, viruses, radiation, and environmental xenochemicals can be immunosuppressive through inducing direct damage or disorganization of the immune system and may also indirectly cause these damage or disorganization by producing ROS. On the basis of animal experiments and observations of cancer incidence in immunosuppressive patients, immunosuppression was first recognized as a factor contributing to carcinogenesis. Immunosuppressors – especially T cell immunosuppressors – may lead to tumor promotion and progression or cocarcinogenic effects. Immunosuppression-induced tumor promotion and progression have been observed in experimental animal models and may combine suppressed cellular immunity with enhanced humoral immunity. It is believed that the cocarcinogenic effect of immunosuppression – thus of environmental immunosuppressors, be they of biologic, physical or chemical origin – mainly concern microorganisms, and particularly oncogenic viruses. Immunosuppressive xenochemicals such as pesticides could be therefore an important etiological factor that may account for the recently observed growing incidence of virus-induced leukemia and lymphoma. Overweight/obesity, type II diabetes, and cancer

Owing to their lipophilicity, many chemical environmental carcinogens can bioaccumulate in the adipose tissue. It has been shown that overweight/obesity can be experimentally induced by benzo[a]pyrene and propose that adipose tissue acts as a reservoir for lipophilic environmental carcinogens, so chemical pollution may in fact generate both overweight/obesity and the accumulation of more chemical carcinogens. Such concept is in addition supported by the fact that epidemiological studies have shown that overweight/obesity is a risk factor associated with certain types of cancer including breast, colorectum and endometrium cancers, and lymphoma. Furthermore, it has been recognized that there is an increased risk of cancer in patients with type II diabetes and that type II diabetes may be induced by chemical environmental pollutants. Therefore, it is proposed that the association of overweight/obesity, type II

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diabetes, and cancer constitute a specific clinical syndrome that comprises environmental diseases and therefore that this syndrome should lead to the search for environmental pollutants as a common cause.

Cancer initiation following disorganization of fetal and neonatal tissues by environmental pollutants

Many clinical studies have revealed the extreme vulnerability of fetus to environmental pollutants, that is, to viruses, radiation, hormones, and xenochemicals. Indeed, the high rate of cell proliferation and differentiation and the lower capacity of metabolic detoxification and DNA reparation render the cells of the fetus and developing child more susceptible to mutations and epigenetic alterations than adult cells. Three periods are individualized during which exposure to environmental carcinogens may take place: the preconceptual period (i.e., effects on parental germ cells); the prenatal period (i.e., exposure of the embryo or fetus via the mother’s placenta); and the postnatal period that corresponds to the direct exposure of children to environmental carcinogens. The enormous complexities of development, which are often especially active at specific times, create fetal and postnatal ‘windows of vulnerability’ to disruption by exogenous factors, during which there may be an increased risk of subsequent development of cancer. This fetal vulnerability exposure window, together with the necessarily prolonged latent promotion phase, may explain why current epidemiological and experimental studies may find no correlation or causation when performed during adulthood. Because there is no protective barrier between the developing fetus and its mother, transplacental exposure of the fetus to natural or synthetic estrogenic hormones and environmental endocrine disrupters can occur and may result in cancer occurrence. Many studies in animal models have confirmed the existence of a causal link between prenatal and neonatal exposure of chemical environmental carcinogens  such as estrogens and endocrine disrupters  and the subsequent development of cancers. From these experiments, an intriguing finding is that tumor promoters such as the ubiquitous estrogenic endocrine disruptor Bisphenol A, administrated during pre- or postnatal periods, can result in the occurrence of prostate cancer or mammary cancer mainly in animal experiments. Perturbations of fetal organogenesis and cell differentiation may therefore be a mechanism whereby exposure to environmental carcinogens could lead to epigenetic alterations and indirect mutagenesis and thus to the subsequent development of cancers, which may occur later in life. Finally, perinatal exposure and even preconceptual paternal exposure to environmental carcinogens might be a causal factor accounting for the

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recently observed growing incidence of some cancer both in children and adults.

Mutagenesis

Cell death

Free radical production as a common molecular effect of carcinogens

As indicated earlier, a common and necessary steplimiting molecular mechanism in carcinogenesis is DNA mutagenesis. Environmental carcinogens may specifically inhibit DNA repair (hence enhance the probability of mutations) and induce genomic destabilization in dividing cells by the means of two mechanisms: a direct inactivation of reparases and the inhibition of expression of repair-associated genes. Two hypotheses have been so far proposed for the second mechanism. According to the first hypothesis, occurrence of a mutator phenotype has been postulated to account for the high rate of nonexpanded (random) mutations observed in cancer cells, and for the genomic instability that may result from this high mutation rate. Such mutator phenotype seems to have been evidenced in early-stage sporadic colorectal cancers, where it might be related to a mismatch repair deficiency. However, to knowledge, no specific gene alteration, as well as no proved causal specific effect of mutagens, have clearly proved that a mutator phenotype is a necessary step limiting in carcinogenesis. Hence, this hypothesis remains to be validated, in as much as according to the classical concept of carcinogenesis cells can acquire mutations and destabilize genome as a result both of a stochastic process associated with clonal (driver) mutation occurrence and the Darwinian selection process acting as a driving force during clonal expansion of mutated cells. As previously mentioned, a cancer-associated molecular mechanism common to many environmental carcinogens might be the production of free radicals. Free radicals are molecules or fragments of molecules containing one or more unpaired electrons that confer a considerable degree of reactivity on these molecular species. Oxidative stress is the cumulative production of ROS and reactive nitrogen species that lead cells to present an unbalanced redox state, conferring advantage of oxidants on reductants. In normal redox conditions, free radicals act as secondary messengers in intracellular signaling cascades, and consequently may contribute at physiological concentrations to tumor promotion. However, as indicated in Figure 1, at intermediate higher concentrations, when the redox potential of the system  that is, the redox buffering capacity of cells  is saturated by an excess of ROS, ROS can damage macromolecules  some of them may further become free radicals  and thus induce oxidative DNA lesions and adducts; although at the highest concentrations they induce cell death.

Tumor promotion

Oxidative stress

ROS

Figure 1 Schematic representation of a dose-dependent hypothetical relationship between oxygen free radicals and cancer genesis during oxidative stress according to Dreher and Junod. Local doses of free radicals capable of cancer genesis are subtoxic. Doses capable of inducing promotion are lower than doses involved in mutagenesis, whereas mutagenic doses, that is, potentially carcinogenic doses are lower than doses inducing cell death.

Conclusion Because all types of environmental carcinogens can generate ROS, it has been proposed that free radicals might be central in carcinogenesis through direct and indirect mutagenesis and promotion induction. As aforementioned, it clearly appears that such presumed carcinogenic effects intimately depend on the intracellular concentration of free radicals. However, in vitro and in vivo tests of intracellular oxidative stress are not sufficient to causally implicate free radicals in mutagenesis and carcinogenesis. It has been observed that many tumor promoters may have a strong inhibitory effect on cellular antioxidant defense mechanisms, but a lack of antioxidants in diet seems to be associated with no more than 5% of overall cancer cases, and administration of antioxidants has not yet been proved to induce preventive anticancer effects. However, in all threatening aspects of life, prevention is better than cure. The most effective anticancer strategy of public officials should be to reduce production of environmental carcinogens.

Further Reading Armitage P and Doll R (1954) The age distribution of cancer and a multistage theory of carcinogenesis. British Journal of Cancer 8: 1--12. Belpomme D, Irigaray P, and Hardell L (2008) Electromagnetic fields as cancer-causing agents. Environmental Research 107: 289--290. Belpomme D, Irigaray P, Hardell L, et al. (2007) The multitude and diversity of environmental carcinogens. Environmental Research 105: 414--429. Belpomme D, Irigaray P, Sasco AJ, et al. (2007) The growing incidence of cancer: Role of lifestyle and screening detection (Review). International Journal of Oncology 30: 1037--1049.

Mechanisms of Environmental Carcinogenesis

Belpomme D, Irigaray P, Ossondo M, et al. (2009) Prostate cancer as an environmental disease: An ecological study in the French Caribbean islands, Martinique and Guadeloupe. International Journal of Oncology 34: 1037--1044. Dreher D and Junod AF (1996) Role of oxygen free radicals in cancer development. European Journal of Cancer 32A: 30--38. Hanahan D and Weinberg RA (2000) The hallmarks of cancer. Cell 100: 57--70. Hinson JA and Roberts DW (1992) Role of covalent and noncovalent interactions in cell toxicity: Effects on proteins. Annual Review of Pharmacology and Toxicology 32: 471--510. Irigaray P, Mejean L, and Laurent F (2005) Behaviour of dioxin in pig adipocytes. Food Chemical and Toxicology 43: 457--460. Irigaray P, Ogier V, Jacquenet S, et al. (2006) Benzo[a]pyrene impairs beta-adrenergic stimulation of adipose tissue lipolysis and causes weight gain in mice. A novel molecular mechanism of toxicity for a common food pollutant. FEBS Journal 273: 1362--1372. Irigaray P, Newby JA, Clapp R, et al. (2007) Lifestyle-related factors and environmental agents causing cancer: An overview. Biomedicine and Pharmacotherapy 61: 640--658. Irigaray P, Newby JA, Lacomme S, and Belpomme D (2007) Overweight/obesity and cancer genesis: More than a biological link. Biomedicine and Pharmacotherapy 61: 665--678. Irigaray P, Lacomme S, Mejean L, and Belpomme D (2009) Ex vivo study of incorporation into adipocytes and lipolysis-inhibition effect of polycyclic aromatic hydrocarbons. Toxicology Letters 187: 35--39.

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Knudson AG (1993) Antioncogenes and human cancer. Proceedings of the National Academy of Sciences 90: 10914--10921. Landau-Ossondo M, Rabia N, Jos-Pelage J, et al. (2009) Why pesticides could be a common cause of prostate and breast cancers in the French Caribbean Island, Martinique. An overview on key mechanisms of pesticide-induced cancer. Biomedicine and Pharmacotherapy 63: 381--395.

Relevant Websites http://www.bioinitiative.org/ BioInitiative Report: A Rationale for a Biologically-based Public Exposure Standard for Electromagnetic Fields (ELF and RF). http://monographs.iarc.fr/ IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. http://www.artac.info The Paris Appeal. International Declaration on diseases due to chemical pollution.