Chemical Carcinogenesis

Question 1

What is the Ames test?

Answer 1

• A test for determining if a chemical is a mutagen - named for its developer, Bruce Ames.
• The use of the Ames test is based on the assumption that any substance that is mutagenic for the bacteria used in his test may also turn out to be a carcinogen; that is, to cause cancer
  
  • Although, in fact, some substances that cause cancer in laboratory animals (dioxin, for example) do not give a positive Ames test (and vice-versa), the ease and low cost of the test make it invaluable for screening substances in our environment for possible carcinogenicity

Question 2

What are the steps and theory behind the Ames test?

Answer 2

• The test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis i.e. it is a auxotrophic mutant, so that they require histidine for growth
• The variable being tested is the mutagen’s ability to cause a reversion to growth on a histidine-free medium
  
  • The tester strains are specially constructed to have both frameshift and point mutations in the genes required to synthesize histidine, which allows for the detection of mutagens acting via different mechanisms
• Some compounds are quite specific, causing reversions in just one or two strains
• The tester strains also carry mutations in the genes responsible for lipopolysaccharide synthesis, making the cell wall of the bacteria more permeable, and in the excision repair system to make the test more sensitive
• Rat liver extract (S9 faction) is added to simulate the effect of metabolism, as some compounds, like benzo[a]pyrene, are not mutagenic themselves but their metabolic products are
• The bacteria are spread on an agar plate with a small amount of histidine
• This small amount of histidine in the growth medium allows the bacteria to grow for an initial time and have the opportunity to mutate
  
  • When the histidine is depleted only bacteria that have mutated to gain the ability to produce its own histidine will survive
• The plate is incubated for 48 hours
• The mutagenicity of a substance is proportional to the number of colonies observed
• The problem of this assay is that Salmonella is a prokaryote and is not a perfect model for humans

Question 3

What is the hepatocyte DNA repair assay?

Answer 3

• Other in vitro assays such as the Hepatocyte DNA repair assay has been developed to more closely mimic human cells
• In this assay, mammalian hepatocyte cells are treated with mutagens to examine DNA damage and repair as demonstrated by the incorporation of radio-labeled 3H-thymidine in the newly synthesized DNA
• This assay can also be extended to whole animal to further mimic the in vivo environment and metabolism

Question 4

What are the stages of chemical carcinogenesis?

Answer 4

• The major stages of chemical carcinogenesis have been deduced over the past ~50 years, primarily from animal model studies (and particularly from studies using the mouse skin model)
• These stages are termed:
  
  • initiation
  • promotion
  • progression

Question 5

What are some important features of the tumor initiation stage of carcinogenesis?

Answer 5

• Tumor initiation begins when DNA in a cell or population of target cells is damaged by exposure to exogenous or endogenous carcinogens leading to mutations in critical target genes
• The responsiveness of initiated cells to their microenvironment gives them a growth advantage relative to normal cells under certain conditions
  
  • In the classic two-stage chemical carcinogenesis system in the mouse skin, a low dose of a carcinogen such as 7,12-dimethylbenz(a)anthracene induces a mutation in Hras1 that does not give rise to tumors over the lifespan of the mouse unless a tumor promoter, such as TPA, is repeatedly applied

Question 6

What are some important features of the tumor promotion stage of carcinogenesis?

Answer 6

• The tumor promotion stage is characterized by selective clonal expansion of the initiated cells, a result of the altered expression of genes
• The products are associated with:
  
  • hyperproliferation
  • tissue remodeling
  • inflammation

Question 7

What are some features of the tumor progression stage of carcinogenesis?

Answer 7

• During tumor progression, preneoplastic cells undergo malignant transformation through a process of selection
  
  • Facilitated by progressive genomic instability and altered gene expression

Question 8

What are some important contributing factors that affect carcinogenesis?

Answer 8

• While the processes involved in each stage of experimental chemical carcinogenesis also appear to be involved in human carcinogenesis, the temporal nature of initiation, promotion, and progression events is more complex
• In addition, multiple mutational events are involved in the formation of human tumors
• Genetic background and nutritional status can dramatically affect susceptibility to a carcinogenic exposure in both experimental animals and humans
• An understanding of the multistage nature of carcinogenesis has led to the discovery of mechanism based inhibitors that target events associated with specific stages

Question 9

What is the process of tumor carcinogenesis in mouse skin?

Answer 9

• In the multistage model of mouse skin carcinogenesis, three mechanistic stages: initiation, promotion, and progression can be defined
• In the initiation stage, mutations occur in critical genes, such as Hras1, which control epidermal proliferation and/or differentiation
  
  • This genetic alteration confers a selective growth advantage on skin epidermal cells such that these cells undergo clonal expansion during the early phase of promotion
• The initiation stage is irreversible and phenotypically silent and the initiated skin behaves as normal skin unless challenged with a promoting stimulus
• The promotion stage of mouse skin carcinogenesis occurs as a result of exposure of the initiated skin to a repetitive promoting stimulus
  
  • Most tumor promoters are not genotoxic but cause altered expression of genes whose products are associated with hyperproliferation, tissue remodeling, and inflammation
  • The endpoint of the promotion stage in the mouse skin model is the formation of squamous papillomas, which are exophytic, premalignant lesions consisting of hyperplastic epidermis folded over a core of stroma
  • Tumor promoting stimuli are very diverse in this model system and include various chemicals such as phorbol esters (e.g., TPA), organic peroxides (e.g., benzoyl peroxide [BzPo]), anthrones such as chrysarobin (Chry), and okadaic acid (OA)
  • In addition, UV light, repeated abrasion, full thickness skin wounding, and certain silica fibers when rubbed on the skin all function as skin tumor promoting stimuli
• The process of tumor progression occurs when papillomas convert into squamous cell carcinomas (SCCs)
  
  • Most, if not all, SCCs that appear during a multistage carcinogenesis protocol in mouse skin arise from preexisting papillomas
  • The SCCs that develop in this model are histologically similar to human SCCs