part 2. mechanisms of carcinogenesis chapter 12. The role of genotoxicity in carcinogenesis David M. DeMarini PART 2 CHAPTER 12 The process of mutagenesis be called DNA-damaging agents. of a molecule between a pair of Instead, it is the cell that produces bases. Again, DNA damage is itself The process of agent-induced mu- the mutation – either through faulty not a mutation and generally does tagenesis consists of three parts: the DNA repair of the mutagen-induced not alter the linear sequence of nu- induction of DNA damage, the sen- or spontaneous DNA damage, or cleotides. A mutation is defined as a sing of the DNA damage by the cell by replicating past the unrepaired change in the sequence or number (the DNA damage response), and DNA damage, thereby introducing a of nucleotides in the DNA. the processing of the DNA damage replication error (Shaughnessy and When DNA damage occurs, the by the cell, which may or may not DeMarini, 2009). cell detects it by means of the DNA result in a mutation. A key under- A description of the process of damage response system and de- lying concept is that mutagenesis is mutagenesis begins with the induc- termines how it will be processed; a cellular process, frequently invol- tion of DNA damage by an endoge- the DNA damage response includes ving DNA replication. Another key nous or exogenous event. Examples DNA repair and apoptosis path- concept is that there is a distinct dif- of DNA damage are DNA adducts ways, which are described in detail ference between DNA damage and (i.e. a molecule bound covalently to by Ciccia and Elledge (2010). The mutation. Thus, mutagens, despite DNA) and single- or double-strand DNA damage response can mediate what their name suggests, generally breaks (i.e. breakage of the phos- the repair of the damage, attempt to do not produce mutations; instead, phodiester backbone). Other types repair the damage but instead pro- mutagens produce DNA damage, of DNA damage are oxidized or frag- cess it into a mutation, or direct the and they might more appropriately mented bases and the intercalation cell to undergo apoptosis. Another Part 2 • Chapter 12. The role of genotoxicity in carcinogenesis 107 possibility is that the damage is not induces mutations in a mutation integrated viral genome (Todaro and repaired at all, and when the cell rep- assay permits it to be classified as Huebner, 1972), and alteration of im- licates, the DNA polymerase correct- mutagenic. munological factors by carcinogens, ly bypasses the damage, resulting in permitting the formation and growth a normal DNA sequence. A brief history of the nexus of tumours (Baldwin, 1973). As time between mutagens and The cell can process DNA dam- has shown, all of the above-men- carcinogens age into three general classes or tioned mechanisms can play a role in the carcinogenic process, espe- types of mutation: gene mutation As reviewed by Claxton et al. (2010), cially in the light of the accumulating (mutations that occur within a gene), there was little direct evidence for evidence for the important role of chromosomal mutation (mutations the role of mutagenesis in carcino- epigenetic mechanisms (Baylin and involving more than one gene, typ- genesis until the early 1970s, and Jones, 2011). ically called chromosomal aberra- before that time only a few carcino- How did the paradigm shift occur tions), and genomic mutation (mu- gens had been shown to be muta- that showed a connection between tations involving the whole genome gens (Burdette, 1955). Indeed, it is mutagenesis and carcinogenesis? – generally aneuploidy, which is surprising to recall that at the time it The first screening studies to test the gain or loss of a whole chromo- was somewhat bold to propose that some). The standard definition of a there was any direct connection be- the hypothesis that some carcino- gens might also be mutagens were gene refers to a segment of DNA that tween the two processes (Miller and performed by Demerec et al. (1951) codes for an mRNA that codes for a Miller, 1971; Knudson, 1973). Many in Escherichia coli and then by protein. The recent Encyclopedia of studies in the 1950s and 1960s Szybalski (1958), who tested more DNA Elements (ENCODE) project showed binding of carcinogens to nu- than 400 compounds in E. coli. indicates that at least 80% of the cleic acids (Wiest and Heidelberger, Although additional testing proceed- human genome is transcriptionally 1953; Brookes and Lawley, 1964). ed throughout the 1960s in a varie- active, but only a small proportion However, before 1972 there was no ty of systems in bacteria, fungi, and of the expressed regions code for direct proof that the electrophilicity mammalian cells, few carcinogens protein (Maurano et al., 2012). of some chemical carcinogens had other than the direct-acting alkylating In the context of the process of a necessary role in the potential mu- tagenic activity of such compounds, agents were found to be mutagens, mutagenesis described above, the or even that DNA, as opposed to leading to the conclusion that carcin- term “mutagen” refers to an agent protein, was the ultimate target of ogens were generally not mutagenic. that can induce DNA damage that carcinogens (Miller, 1970). However, this view began to shift the cell processes into a mutation. Although sound theoretical rea- when Malling (1966) combined a The more general term “genotox- sons had been proposed to support chemical hydroxylating mixture with in” refers to an agent that induces the notion that carcinogens might act the carcinogens diethylnitrosamine DNA damage that may or may not through a mutagenic mechanism, and dimethylnitrosamine, which be processed by the cell into a mu- a clear demonstration of this con- were not mutagenic in vitro, and tation. Some assays for genotoxici- nection did not yet exist (Miller and showed that the resulting metabo- ty, for example, measure only DNA Miller, 1971). Thus, binding to DNA of lites were mutagenic in the fungus 32 damage, such as P-postlabelling metabolites of carcinogens had been Neurospora crassa. Malling (1971) and the comet assay, whereas oth- identified, but there were no data to then prepared an enzymatic activa- er assays measure mutation, such show that these DNA adducts were tion system composed of the super- as the Salmonella typhimurium re- processed into mutations or that mu- natant from mouse liver homogenate verse mutation test, the Hprt gene tations themselves played a role in centrifuged at 30 000g (microsomes) mutation assay in Chinese hamster carcinogenesis. Consequently, mu- plus cofactors, and showed that di- ovary cells, and transgenic mouse tagenesis was viewed at that time as methylnitrosamine was mutagenic mutation assays. Thus, finding that an equally plausible mechanism for in S. typhimurium in a liquid suspen- an agent induces DNA damage carcinogenesis, along with epigenet- sion assay in the presence of this ac- would permit it to be called genotox- ic changes (Miller, 1970; Miller and tivation mixture. Additional evidence ic, and showing that the agent also Miller, 1971), altered expression of an that carcinogens could be mutagens 108 after mammalian metabolism was Indeed, a comprehensive analysis present at high frequencies in tu- provided by Legator and Malling showed that more than 90% of the mours, DNA sequencing methods (1971) with the host-mediated assay. IARC Group 1 chemical carcinogens were introduced in 1977 (Pettersson Ames et al. (1972) introduced the are genotoxic (Waters et al., 1999). et al., 2009), which provided the tech- use of the plate incorporation assay The current genetic toxicity test nical means to directly determine the in Salmonella and demonstrated that battery is based on this relationship presence and types of mutations in DNA-reactive metabolites of known between mutagenesis and carcino- any gene or chromosome. carcinogens were direct-acting mu- genesis. Consequently, mutagenic- DNA sequencing of mutations tagens. The connection between ity assays continue to be used as induced in selected genes by a lim- mutagenesis and carcinogenesis a potential screen for carcinogens, ited number of mutagenic carcino- was extended when Ames et al. and the results are used for regula- gens in microbes in the 1980s and in (1973) combined a rat liver homoge- tory purposes throughout the world mammalian cells and tumours in the nate centrifuged at 9000g (S9 frac- (Eastmond et al., 2009). For exam- 1990s began to show that any par- tion) plus cofactors prepared as de- ple, a positive result in the Salmonella ticular mutagen produced an array scribed by Garner et al. (1972) with mutagenicity assay indicates a 70% of mutations and that these varied Salmonella and a variety of rodent probability that the test chemical is among the genes and cells exam- carcinogens then considered to be a rodent carcinogen (Zeiger, 1998). ined. A variety of mutagens produce non-mutagenic in the plate incorpo- When a randomly selected set of similar mutation spectra, and the PART 2 ration assay and showed that these 100 organic compounds was tested predominant base substitution that CHAPTER 12 carcinogens were, in fact, muta- in the Salmonella mutagenicity as- an agent induces in one system genic. Additional refinements of the say, about 20% of them were posi- is generally the same one that the Salmonella tester strains and the tive (Zeiger and Margolin, 2000). agent produces predominantly in all conduct of multiple testing studies, Thus, out of an estimated 80 000 other systems across the phyloge- involving not only Salmonella but such compounds in commercial use, netic scale, from bacteria to humans also other test systems (Tennant 16 000 (20%) may be positive for (DeMarini, 1998, 2000). Thus, in et al., 1987), resulted in the current mutagenicity in the Salmonella mu- terms of the predominant base sub- recognition that many carcinogens, tagenicity assay, and 11 200 (70%) stitution produced by agents, there is by themselves or after metabolic of those may be potential rodent concordance across species in that activation, are mutagens, and that carcinogens.