The Cellular Response to the Genotoxic Insult

The Question of Threshold for Genotoxic Carcinogens

Helmut Greim, Richard Albertini, Helmut Greim, Richard Albertini

• Publisher: Royal Society of Chemistry
• ISBN:
  9781782626046
• Edition: 1
• Topic: Scienze biologiche
• Subtopic: Biochimica

Genotoxic carcinogens can lead to DNA mutations with the potential to cause cancer. Typically, a series of mutation events are needed before malignancy occurs so a single, small exposure may not result in disease. Also, cells have an armoury of defence mechanisms which, to a degree, counter the effects of mutagens. Distinguishing the point at which exposure to a carcinogen increases mutation rates beyond the background level is challenging. In fact, there is now general agreement that, for genotoxic carcinogens, no specific threshold can be identified. However, NOAELs (No Observed Adverse Effect Levels) may be used in the process of establishing a dose-response relationship. These denote the level of exposure at which there is no significant increase in adverse effects in the exposed population when compared to an appropriate control. Such a scientifically defendable threshold allows us to propose health based exposure limits for genotoxic carcinogens. This book describes the various cellular defence mechanisms individually and explains how they are regulated. The processes covered include metabolic inactivation, epigenetic regulation, scavenging mechanisms, DNA-repair and apoptosis. It also considers dose-dependent threshold mechanisms of carcinogenesis and the rate limiting parameters. Aimed at graduate level and above, the book discusses the consequences of genotoxic evaluation and urges readers to question the idea that even low exposures present a cancer risk.

1. Threshold Effects Observed in Experimental Studies

CHAPTER 1.1

Mechanisms Responsible for the Chromosome and Gene Mutations Driving Carcinogenesis: Implications for Dose-Response Characteristics of Mutagenic Carcinogens

R. JULIAN PRESTON

National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711, USA

Email: [email protected]

Through the use of ultra high throughput DNA sequencing techniques, it has been possible to characterize a number of tumour types at the molecular level. This has led to the concept that there are ‘driver’ and ‘passenger’ mutations, with an estimate of the number of driver mutations being about 120 for all tumours characterized to date. Furthermore, it is proposed that for any particular tumour a subset of these driver mutations results in the development of the phenotypes underlying the six acquired characteristics defined by Hanahan and Weinberg in their Hallmarks of Cancer. In the framework of risk assessment, these acquired characteristics are key events along the pathway from a normal cell to a metastatic tumour that can be induced by DNA-reactive carcinogens. The nature of the dose–response curve for the driver mutations is influenced by the mechanism of induction of these mutations. In general, mutations (both gene and chromosomal) induced by DNA-reactive carcinogens are formed by replication errors on a damaged DNA template (i.e. DNA adducts). By considering such chemically induced mutations in the context of classical ‘hit’ theory, one hit is required to produce a gene mutation and two independent hits are required to produce a chromosomal mutation. The consequence is that the dose–response curve for gene mutations is linear and that for chromosome mutations is proportional to the square of the dose. Thus, for a typical DNA-reactive carcinogen that induces both gene and chromosomal mutations, the dose–response curve for total mutations will be a linear–quadratic with the form being influenced by the relative proportions of gene and chromosome mutations.

1.1.1 Introduction

Over the past few years the development of sophisticated techniques for sequencing genomes and for establishing gene expression profiles, for example, has led to a much more detailed characterization of tumors at the molecular level.¹ What is apparent is that the carcinogenic process is complex and involves a range of interacting genotypic and phenotypic alterations to drive a normal cell to a transformed one and ultimately to a tumor. In addition, the role of environmental agents in initiating or accelerating this process is becoming better understood. However, putting all of these types of information together to predict the likelihood of an exposure scenario inducing tumors above the background level is really a daunting task. This is necessary if it is going to be possible to characterize tumor dose-response curves in a qualitative and quantitative manner. The aim of this chapter is to consider the form of tumor dose-response curves for mutagenic chemicals and to determine whether or not there is the potential for non-linearity or perhaps a threshold response.

1.1.2 Genetic Alterations and Cancer

It is well-recognized that all cancers contain somatic mutations. In fact, it has been shown that a metastatic tumor contains in excess of 10 000 genetic alterations, both gene and chromosomal.² However, it has been determined that a subset of these alterations can be considered to be driver mutations because they confer a selective growth advantage and thus are integral to cancer development.³ The non-driver genetic alterations are termed ‘passengers’ because they are a product of the cancer process that confers genomic instability on cells.

Recent studies have shed some light on the drivers for specific tumors and for cancers overall. Pleasance et al.³ sequenced the genomes of a malignant melanoma and a lymphoblastoid cell line from one individual and thereby developed a catalogue of the somatic mutations from an individual cancer. Although the specific drivers could not be identified from a single cancer, the method could be applied to a large number of similar cancer types to identify the drivers. However, it was possible to identify mutations that were consistent with exposure to ultraviolet (UV) light—a known risk factor for melanoma.

Using a similar ultra high throughput sequencing approach, Greenman et al.¹ re-sequenced 210 cancer cell genomes and concluded, based on computational analysis, that there were driver mutations in about 120 genes, with the remainder (of the order of a thousand) being passengers. Based on this study, it needs to be noted that when developing models of cancer probability from environmental exposures leading to considerations of dose-response, the genetic ‘target’ should be considered to be no larger than this set of driver mutations.

Along similar lines, Stephens et al.⁴ studied another major class of genetic alteration associated with cancer formation, namely somatic rearrangement. These investigators used a paired-end sequencing strategy to identify somatic rearrangements in breast cancer genomes. Such rearrangements were more frequent than anticipated and were largely intrachromosomal. This study exemplifies the role of chromosomal alterations in cancer formation. Bignell et al.⁵ characterized homozygous deletions in cancer cells and concluded that many are in regions of inherent fragility, but that just a small subset overlies recessive cancer genes.

These two studies highlight the involvement of structural chromosome changes in the cancer process and demonstrate that they can also consist effectively of drivers and passengers. It remains uncertain whether or not genetic alterations in driver genes are produced by the same mechanism(s) as passenger alterations. For example, driver mutations as tumor inducers are more likely to be induced by the exposure, whereas passenger mutations are quite likely to be produced through changes in cellular housekeeping processes as a consequence of driver mutations. This could have an impact on dose-response characterization for tumors, especially for mutagenic chemicals. This issue of mechanisms of formation of genetic alterations will be returned to later in this chapter when discussions are introduced of how mechanism affects dose-response curve shape.

1.1.3 Carcinogenic Process

The carcinogenic process has for a long time been considered to be readily divided into three discrete phases: initiation, promotion and progression.⁶ For mutagenic chemicals, it can be considered that initiation is driven by induced mutation and that promotion and progression are under the control of processes that are independent of the mutagenic exposure—perhaps other chronic exposures or endogenous processes. On the other hand, all three stages could be enhanced by chronic exposures to mutagenic chemicals (or mixtures) because the exposure extends over a time frame that is consistent with the extended time for a tumor to develop. The requirement for the latter is that exposure has to be extended over years (for humans), whereas in the case of initiation, exposure to the mutagenic agent could be acute or very short term. Such differences can have significant effects on dose-response considerations since the different exposure scenarios can have quite different effectiveness for inducing genetic alterations.

An alternate description of carcinogenesis is provided by Hanahan and Weinberg⁷ in their 2000 paper, Hallmarks of Cancer. From considerations of some characteristics of a broad range of tumor types for different target tissues and in several different species, Hanahan and Weinberg described a set of six acquired characteristics essential to transform cells and for the development of a metastatic tumor. These characteristics are:

• unlimited replicative potential;
• ability to develop blood vessels (angiogenesis);
• evasion of programmed cell death (apoptosis);
• self-sufficiency in growth signal...