Carcinogens, DNA Damage and Cancer Risk

The following sections are included:

• Preface
• Contents

Many cancers caused by chemical carcinogens do so through a mutagenic mechanism initiated by the formation of carcinogen–DNA damage. Some chemical carcinogens can damage DNA with no metabolic intervention, and others must be biotransformed. DNA adduct formation is considered necessary but not sufficient for tumorigenesis, as dosimetry for DNA adducts and tumorigenesis has been amply demonstrated in experimental models. Once methods became available to measure DNA adducts in humans, studies showed widespread distribution of DNA adducts in human tissues, dosimetry for DNA adduct formation in circumstances where exposure was accurately known, and consistent positive associations between DNA adduct formation and human cancer risk. Case–control and nested case–control epidemiological studies, examining DNA adducts in individuals exposed to a single carcinogen or carcinogenic mixtures, have shown, for many cancers, that individuals with the highest DNA adduct levels are also those with the highest cancer risk. The modest nature of these associations (odds ratio or relative risk between 1.25 and 9.10) indicates that, although DNA damage is essential, other events typically contribute to human cancer etiology.

This chapter summarizes the latest data increasing our knowledge on a function of cytochrome P450 (P450) enzymes in metabolism of several carcinogens and on the mechanisms of the reaction cycle catalyzed by the P450-monooxygenase system. Particularly it describes the P450- dependent metabolism, toxicity, and carcinogenicity of two human environmental carcinogens, benzo[a]pyrene (BP) and aristolochic acid I (AAI). This chapter focuses on studies that have specifically investigated the role of P450 enzymes in the bioactivation and detoxification of BP and AAI, and the lessons learned from these studies in examining the potential role of P450-mediated metabolism for other known or putative human environmental carcinogens. P450 enzymes play a key role both in metabolic activation of these human carcinogens to the reactive intermediates forming DNA adducts, and in their detoxification to metabolites that are excreted. While BaP is oxidatively metabolized (activated or detoxified) by P450s, AAI can be metabolized both oxidatively (to its detoxification metabolite aristolochic acid Ia [AAIa]) and reductively (to be activated to DNA-binding metabolites). Concerning the mechanisms of reactions catalyzed by the microsomal P450 monooxygenase system reviewed in this chapter, it is demonstrated that besides NADPH:cytochrome P450 oxidoreductase (POR), the system consisting of microsomal NADH:cytochrome b₅ reductase together with its substrate cytochrome b₅ and cofactor NADH can function as a donor of electrons both for the first and the second reduction of at least some of P450 enzymes in their reaction cycle. These findings indicate that the old myth, the generally accepted mechanism on the exclusivity of the POR/NADPH system for reduction of several P450s, should be revised.

DNA damage is the critical step linking carcinogen exposure from tobacco products to cancer-causing mutations in important growth control genes. This chapter provides an overview of carcinogens in tobacco products and their conversion to metabolites that react readily with DNA bases producing addition products known as DNA adducts found in smokers’ DNA. DNA adducts can cause errors during DNA replication leading to permanent changes in DNA sequence. When such mutations occur in critical regions of oncogenes such as KRAS and tumor suppressor genes such as p53, the result can be loss of normal growth control mechanisms and cancer development. This chapter presents evidence for this important biochemical pathway.

Polyclonal and monoclonal antibodies have been developed that recognize the guanine adducts formed by the reactive intermediates of benzo(a)pyrene, a ubiquitous environmental carcinogen. The antibodies cross-react with the guanine adducts of several other polycyclic aromatic hydrocarbons (PAH) and thus can detect a wide range of PAH diol epoxide guanine adducts. These antibodies have been used in highly sensitive enzyme-linked immunosorbent and immunohistochemical assays to monitor human exposure to PAHs. Occupational, lifestyle, and environmental exposures to PAHs have been detectable by measurement of white blood cell DNA adduct levels. Other studies have used the assays to investigate the role of PAHs in cancer induction. In the last 30 years, data produced using antibody-based methods have been essential for much of the progress in our understanding of the hazards of PAHs.

Currently lung cancer accounts for 1.7 million deaths worldwide and more than 150,000 deaths in the United States yearly. The molecular events responsible for the development of lung carcinogenesis are becoming elucidated with continuing research. Carcinogens in tobacco, tobacco smoke, vehicle exhaust, and industrial effluents are inhaled mostly and act either directly or following biotransformation to damage DNA. DNA damage leads to dysregulation of cell growth and renewal, which in turn is followed by mutagenesis, hyperplasia, and malignancy. This chapter describes the multistage process of human chemical carcinogenesis through the example of lung cancer. Major players in the carcinogenic process leading to human lung cancer include chemicals of the classes: (1) polycyclic aromatic hydrocarbons (PAHs), (2) tobacco-specific nitrosamines (TSNs), and (3) diesel engine exhaust fumes (DEEF). Evidence of exposure to these classes of chemicals can be found through DNA adduct formation in normal lung tissue, mutational spectra in lung tumor DNA, and changes in gene expression specific to lung tumor pathology. Knowledge of the dysregulated biological pathways involved in carcinogenesis, including proliferative mechanisms, tyrosine kinase upregulation, motility, differentiation, angiogenesis, and metastasis, have led to development of individualized or precision therapies. In the future, early detection of lung cancer will likely involve low-dose computed tomography, which will allow successful implementation of personized therapeutic approaches.

Since its discovery over 50 years ago aflatoxin has been shown to be one of the most potent environmental carcinogens in experimental models and in epidemiologic studies. It is linked to the development of liver cancer and other health problems in people. Exposure to this carcinogen from the diet varies enormously across the world and many human studies have employed aflatoxin specific biomarkers to characterize health risks from these exposures. The development of these biomarkers has been predicated upon mechanistic investigations that has identified and characterized the DNA and protein adducts formed by the metabolism of this compound. In turn these biomarkers have also proven to have efficacy for the measurement of successful primary prevention strategies and chemoprevention technologies. Thus, the translation of mechanistic studies of aflatoxin in experimental models to both epidemiologic investigations in high-risk populations in Asia and Africa and the use of these markers to design public health prevention methods makes aflatoxin a paradigm for other environmental carcinogens.

Many metal compounds are classified by many agencies around the world as known or probable human carcinogens. Occupational and environmental exposure to metals are risk factors for both workers and the general population. However, mechanisms for metal induced carcinogenesis are poorly understood. Metals can act as mimics for essential elements, increase free radicals or disrupt protein structure and folding. The physical and chemical properties of metals can also affect their potency and bioavailability. Here we use chromium as a model metal carcinogen to address the potential mechanisms of its carcinogenicity. We discuss the cellular uptake and intracellular reduction of hexavalent chromium, its effect on DNA repair, its effect on cell cycle and mitosis, and epigenetic alterations including methylation, histone modification and microRNAs.

A number of important cancer chemotherapeutic drugs, best represented by those belonging to the groups of platinum drugs, nitrogen mustards, nitrosoureas and methylating agents, bring about their therapeutic effects by forming with the DNA of cancer cells covalent complexes (DNA adducts) which block cell replication and thus lead to cell death. The amount of such adducts accumulating in the cells of a given patient represents the biologically relevant dose of drug reaching the patient’s tissues and, as such, may constitute a marker susceptibility to the drug’s therapeutic effects at the individual level. Based on this anticipation, extensive efforts have been made to develop analytical methodologies for measuring drug-DNA adducts and to apply them in studies aimed at the evaluation of the relationship between DNA adduct accumulation and patient therapeutic response.

The great majority of such studies have focused on the categories of drugs mentioned above, and have utilized a range of analytical methodologies, including immunochemical assays, liquid chromatography with different modes of detection including mass spectrometry, and molecular biology-based methods. Although cancer tissues would be the ideal targets in which adducts should be measured, the great majority of studies have utilized blood leukocytes as surrogate tissues in view of their ease of accessibility. Almost all reported studies have been of relatively small size, a limitation whose consequences are compounded by the complexity imposed by the clinical settings in terms of the variability of treatment protocols (use of a single drug or drug cocktails, different dosages and modes of administration, different types of cancer and sub-types of tumor, etc.). As a result, the degree of success of the reported studies, i.e. the observed correlations between adduct accumulation and patient response to treatment, varies between different drugs and different studies. However, despite these complications, overall clear evidence has accumulated that patients who accumulate higher levels of drug-DNA adducts, even in surrogate tissues like blood cells, have a higher probability of a positive response. Overcoming the difficulties mentioned and achieving further progress in this field would require studies on a much larger scale, which can be achieved through multi-center collaborations, and ideally combining measurements of adduct accumulation with downstream biological responses.

Nucleoside analogs were first approved by the U.S. Food and Drug Administration for use against the human immunodeficiency virus-1 (HIV-1) in 1987. Since then, these agents, now commonly referred to as nucleoside reverse transcriptase inhibitors (NRTIs), have become essential components of the highly active antiretroviral therapy (HAART) drug combinations used for treatment of HIV-1 infections. Incorporation of the drug into host nuclear (nDNA) and mitochondrial DNA (mtDNA) may be largely responsible for dose-limiting toxicities. Zidovudine (AZT, 3′-azido-3′-deoxythymidine), the first NRTI approved for the therapy of HIV-1, is incorporated into DNA, causing mutations, and inducing micronuclei (MN), chromosomal aberrations, sister chromatid exchanges, shortened telomeres, and aneuploidy in cultured cells. Genomic instability would be predicted as a consequence of these events. Animal models, such as mice and monkeys, as well as ex vivo models, such as human placental perfusion, have provided insights into our understanding of AZT-induced genotoxicity, and are discussed here. Detailed information on the mechanisms underlying NRTI-associated antiretroviral efficacy, toxicity, and metabolic resistance were not available when AZT was first approved for use as an antiretroviral agent. Our increase in knowledge, acquired through these models and observations in the clinic, may lead to intervention strategies to attenuate toxicity without altering drug efficacy.

Most environmental carcinogens produce DNA damage that leads to mutational events in important cellular genes. Ultimately, the accumulation of mutations in multiple genes leads to loss of growth control and cancer development. Investigations over the past several decades have resulted in the identification of numerous naturally-occurring and synthetic compounds that exhibit the ability to inhibit the development of cancer by a variety of mechanisms. The ability to reduce DNA damage and ensuing mutational events associated with this damage is an important role for these compounds, generally referred to as chemopreventive agents. This chapter discusses mechanisms by which chemopreventive agents reduce DNA damage and adduct formation using specific compounds and vitamins as examples. The principal criteria for selecting these compounds for discussion is the fact that data were available on the mechanisms by which they reduce not only DNA damage but also tumorigenesis in specific organs. Protocols for the identification of agents that reduce DNA damage in animal model systems are given and some results from human clinical trials are presented. It is important to recognize that there may be many naturally-occurring chemopreventive agents that have not as yet been identified and many of them undoubtedly have the ability to reduce DNA damage. Probably the most effective agents are those that have a dual role of reducing DNA damage and mutational events as well as cellular and molecular events associated with tumor promotion/progression.

The risk of the cancer development in humans is determined by the interaction of two main cancer-causing factors, genetics and carcinogen exposure. It is well-established that exposure to chemical carcinogens may trigger the carcinogenic process by affecting the genome, through DNA damage and mutations, and epigenome. Through examples of the accumulation of carcinogen-induced DNA damage and epigenetic alterations caused by genotoxic and nongenotoxic chemicals, this chapter summarizes current knowledge related to the role of DNA damage and epigenetic abnormalities as drivers of the carcinogenic process. It also highlights and emphasizes a tight link and mechanistic interconnectivity between DNA damage and epigenetic alterations in carcinogenesis.

DNA and protein adducts are validated biomarkers of human exposure to carcinogens and, in some instances, of risk of developing cancer. As derivatives of reactive chemicals, adducts may also provide unique mechanistic insights. Until recently studies have adopted a “bottom-up” approach, whereby exposure to known or suspected agents is monitored. The advent of the “exposome (see Glossary)” concept, which seeks to define the totality of the influence of the environment on disease susceptibility, has led to a more untargeted top-down “adductomics” approach to identifying multiple modifications to DNA or protein (hemoglobin and albumin), attempting to capture entire populations of adducts. Although still in its infancy, the approach offers the prospect, particularly when combined with other omic approaches, such as proteomics, transcriptomics and metabolomics, all of which have potential for highthroughput screening of populations, of capturing snapshots of human exposure to genotoxicants and the resultant biological consequences at multiple stages throughout life. This should shed significant light on the causes and courses of chronic diseases afflicting humans and provide new opportunities for early interventions for disease prevention.

The following section is included:

• Index