1. Chapter 5
Chemicals and Cancer

2. Aniline Dye and Cancer
Extraction of aniline from Coal was the beginning of the aniline dye industry.
Later 2-naphthylamine was used as the starting material for this process.
Factory workers developed bladder cancer.
Aniline extracted from coal tar and oxidized with potassium hydroxide….it formed a dark brown precipitate.
-When alcohol is added to the dark sludge, aniline dye was generated. It is the first synthetic dye.
-Later 2-naphthylamine became an ideal starting material for the synthesis of many dyes…..and mass production in Germany in 1890.
-Increase in bladder cancer was observed in these factory workers. There is both epidemiological and experimental evidence to suppor tthis.
-This was the first example of a chemical causing human cancer.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

3. Chemical Carcinogenesis
Principle of Chemically induced Carcinogenesis:
Long Delay
Dose Dependence
Organ specificity
Examples of Chemical Carcinogenesis
Asbestos and Mesothelioma
Pesticide, ethylene dibromide (EDB)
Dioxin
Identification of 2-Naphthalamine as a carcinogen led to the identification of several principles about carcinogens.
-Principles:
-Long delay…..factory workers did not begin developing bladder cancer until about 10 years after the initial exposure to the carcinogen, with most cases taking btw years to develop. This is indicative of the multi-step process of cancer progression.
-Dose Dependence – individuals exposed to 2-Naphthalmine for longer periods of time (had greater exposure to the chemical) had higher incidence of developing bladder cancer.
-Organ specificity – for this carcinogen bladder is the primary target. This is typically casued by the selective ways in which chemicals make contact or accumulate in certain body tissues. For example, chemicals that get concentrated in the urine tend to cause bladder cancer, while chemicals that are inhaled tend to cause lung cancer.
There are numerous examples of chemical carcinogens and the cancer that they cause.

4. Asbestos and Mesothelioma
Asbestos is a great example of the organ specific nature of carcinogens. Asbestos became widely used in the late 1800s due to its fire retardant properties……oven mittens, fire proof clothing and shingles for houses.
-When asbestos breaks down it forms a fine dust that contains numerous sharp needle like fibers that are so tiny that you can only see them under an electron microscope. These needles can be inhaled and lodge in the lungs where they cause scarring that kills people through suffocation. This disease is called asbestosis. This first cases of which were identified in the 1920s.
-Shortly after this cases of lung cancer also began to increase in individuals exposed to asbestos particles.
-Asbestos also has the ability to cause mesothelioma…..a rare cancer of the mesothelial cells that cover the interior surfaces of the chest and abdominal cavity. A dramatic increase in the number of cases of mesothelioma was observed in asbestos workers
-Mechanism: Microscopic fibers of asbestos become embedded in the lung and gradually penetrate thorugh the lung tissue and emerge into the chest cavity. In this location the asbestos fibers trigger chronic irritation and inflammation that promotes the development of cancer in the mesothelial cells that covers the lungs and lines the interior chest wall.
-Now of course there is governmental regulation of asbestos….so the incidence of asbestos related cancers should begin to decline over the next 20 years.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

5. Industrial Pollution and Cancer
Low doses of carcinogens released into the environment.
Cancer risk is related to cancer dose.
Historical trends do not support the argument for industrial pollution.
Cancers that arise in the workplace are typically triggered by sustained high dose exposures to specific carcinogens. Small amounts of the same chemicals may be released into the environment….contaminating the air we breathe or water that we drink.
As such, there is always a lot of talk about the relationship of industrial polluttion and cancer. If you take a look here….you can see the point at which industrial pollutants began to dramatically increase, but you see no real change in the number of breast or colon cancer cases for the next 40 years or so.
-What are the reasons that this is not true?
-Cancer risk is related to cancer dose…..general public is exposed to environmental chemicals that are orders of magnitude less than that of factory worker. Such low conc. of chemicals are not very carcinogenic.
-Historical trends of cancer do not support increased environment pollutants. You would have expected to see a huge boom in the number of cancer cases in the 20th century due to the explosive growth of industrial chemicals that has occurred between s. You would have expected that any cancer epidemic would have emerged by now. This is shown in this figure. The only cancer to increase is lung cancer which we know is assoc. with the increase in cigarette smoking, not industrial pollution.
-this tells you that industrial pollution has little negative environmental impact.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

6. Low Dose Environmental Pollution
The epidemiological approach is not sensitive enough to detect small increased in cancer risk.
Limitations of Animal Models in assessing low dose carcinogen exposure
Maximum Tolerated Dose
Threshold
Since epidemiclogical data is not usually useful for detecting small increases ( anything less than a doubling) in cancer incidence. In other words it is probably not good for detecting low dose carcinogen exposures, scientists usually turn to animal testing to address their role in cancer.
-However, animals testing also has certain limitations:
-One problem is the need to obtain a big enough number of cancer cases to generate statistically reliable results. To deal with this animals are typically treated the maxium tolerated dose (amount of the potential carcinogen that is equivalent to the highest dose possible without cause immediate life-threatening consequences). These high doses cause dramatic tissue destruction causing the remaining cells to rapidly proliferate to try an replace the damaged cells. As a result there is an increased chance that proliferation of a cell containing damaged DNA will occur and cancer will result. Why is this a problem…..well it is the substance really carcinogenic are just toxic???
-If the cancer results from the ability of the substance to cause a high rate of cell death and not that the substance is inherently mutagenic, they you would predict that lower doses that do not kill cells would also not cause cancer. In this particular case, then the dose response curve for this substance would show a threshold. In other words, that a certain dose must be exceeded before cancer rates increase.
-This figure illustrates why threshold can be a problem with animals data. It shows data from exposure to benza[a]pyrene, a carcinogen present in gasoline exhaust fumes and smoke generated by burning organic matter. The graph on the left is data collected from a series of dose treaments…..you might then think that you can extapolate that graph to determine the effect of low doses of the chemical. However, if that data were actually collected you would see that there is not a linear relationship between dose and cancer risk.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

7. High and Low Dose Cancer Risk
Linear Model
Threshold Model
Hormetic Model
Extrapolation of animal Data
Known human carcinogens
Reasonable anticipated human carcinogens
Because of these issues, cancer biologist typically have one of three ways to deal with understanding the relationship between high and low dose cancer risks.
-Linear Model which assumes that there is a linear relationship between dose and cancer risk.
-Threshold model that assume there is not cancer risk at doses lower than the threshold and a linear dose response relationship after the threshold.
-These are dose response curves that are U shaped. This is called hormesis and it reflects a decline in cancer rates at very low doses of carcinogen exposure and increases as the dose increases. One example of this is dioxin. In a way, this model also includes a thresholdl.
-The concept of threshold may be related to the time it takes for a substance to cause tissue destruction and the resulting stimulation of proliferation of the remaining cells. Another possibilty may be that at low doses, cells may be able to repair the DNA damage caused by the carcinogen, but at higher doses the cell’s repair mechanism just cannot keep up.
-One additional point to mention here is that not all carcinogenic agent have equivalent risk to animals and human. Saccharin caused bladder cancer in rats, but its peak use was in the 1970s and we have seen no association of bladder cancer and saccharina usage in humans. Since then it has been shown that when rats ingest ssodium saccharin, a crystalline precipitate forms in the bladder. This irritates the lining of the bladder which in turn stimulates cell proliferation and increaseing cancer risk of those animals. The most important point is that this precipate only forms when there is proteins in the urine and rats have a fold higher protein conc. In their urine than humans do.
-Known human carcingens—Aflatoxins, Hep B virus, Mustard Gass, Tobacco, UV radiation, Benzene, Nickel Compounds, Radon
-Reasonable anticipated human carcinogens –Acrylamide, Nitrobenzene, polycyclic aromatic hydrocarbons, Cisplatin, Chloroform
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

8. Categories of Chemical Carcinogens
Now that we have talked about the relationship of chemicals and cancer, let’s begin to think about the mechanisms used by these various chemicals to cause cancer. There are several different mechansims based on the category of chemical carcingen. So lets first take a look at the various categories of chemicals carcinogens.
-Most chemical carcinogens are organic chemical (carbon containing). They range in size from small to very large complex organic molecules.
-Polycyclic aromatic hydrocarbons- a diverse group of compounds constructed from mutliple fused benzene rings. They are natural products of coal tars, soots and oils and theya re also produced by the incomplete combustion of coal(grilling). The carcinogenic potency varies from weak to very potent.
-Aromatic Amines – these are organic molecular that possess an amino group (NH2) attached to a carbon backbone containing one or more carbon rings. Many of the compounds in this group were once employed in the manufacturing industry. These substances vary from stongly carcinogenic to those that are not carcinogenic at all.
=N-nitroso compounds – are organic chemicals that contain a nitroso group (N=O) joined to a nitrogen atom. Most of these compounds are industriral or research chemicals, although a few of them are encountered in cigarette smoke. In addition, nitrates and nitrites found in cured meats can also be converted in the stomach to nitrosamines. However, none of these compounds have been directly linked to human cancer.
-Alkylating Agents –these are molecular that readily undergo reactions in which they attach a carbon-containing chemical group to some other molecule. This group of chemicals is defined by their chemical reactivity (no structural features) or their ability to join a chemical group to another molecule. One example of this is vinyl chlordied.
-natural products – theseare a structurally diverse group of cancer causing molecules produced by biological organisms mainly microorgansims and plants…..things such as aflatoxin made by the mold aspergillus.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

9. Chemical Carcinogens
All of these chemical as considered carcinogens b/c they cause cancer in humans or animals. However, this designation does not mean that they directly trigger cancer.
-As I mentioned before, carcinogens trigger cancer through a variety of mechanisms.
-Take 2-napththylamine that causes bladder cancer in industrial workers, but rarely causes cancer when injected into the bladder of animals. This is because when it is ingested or inhaled by humans it passes through the liver where it is metabolized into a chemical compound that does caused cancer. This process is referred to as metabolic activation and the substances that behave in this manner are called precarcinogens.
-This type of activation of the precarcinogens is carried out by liver proteins that are members of the cytochrome p450 enzyme family. These enzymes catalyxe the oxidation of ingested foreign chmicals .
-This figure shows a hydroxylation reaction which is one of the ways oxidation reactions can be catalyzed by p450 to make chemicals more water soluble so that they can be excreted in the urine.
-Sometimes this process results in conversion of the substances into carcinogens
-This explains the rational for using liver extract in the AMES test.
-There is lots of evidence to support the importance of p450. Mutant mice that produce an abnormal amount of cytochrome p450 1A1, a form of p450 that oxidizes polycyclic hydrocarbons show an elevated rate of cancer. Also, when p450 was inactivated, the cancer rate in these animals was diminished.
-Some people inherit a form of cytochrome p450 1A1 that is produced in excess in response to tobacco smoke. So if this person smokes, then he/she has an even higher risk of developing lung cancer than other smokers.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

10. Electrophillic Carcinogens
When carcinogens are metabolize in the liver, they can be converted to highly unstable compounds containing electro deficient atoms. These molecules are said to be electrophillic or electron loving. These molecules will readily interact with substances that are rich in electrons.
-DNA, RNA and proteins are all electron rich atoms……so they become a good target for electrophillic carcinogens. DNA of course is the prime target b/c carcinogens cause DNA mutations.
-this figure shows the result of an experiment done to test this. Here animals were injected with various polycyclic hydrocarbons that differed in carcinogenic potency. Cells were isolated from the treated animals and measurements were made to determine with intracellular molecules had become bound to the polycyclic hydrocarbons. The data shows a direct relationship between the carcinogenci potency of different polycyclic hydrocarbons and their ability to become covalently linked to DNA. The ones with greatest DNA binding ability caused the most cancer.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

11. Electrophillic Carcinogens
However, before a polycyclic hydrocarbon can interact with DNA it must be activated.
-Benzo[a]pyrene is normally a nonreactive, nonmutagenic compound. When it is ingested and metabolized by the liver by cytochrome p450 it is activated to a derivatives containing an epoxide group.
-What is an epoxide? It is a 3 membered ring containing an oxygen atom covalently bonded to 2 carbon atoms that are electron deficient. As a result they tend to react with electron rich molecules like the amino nitrogen found in the DNA base, guanine. This causes the benzo[a]pyrene to become covalently bonded to DNA, forming a DNA adduct (DNA-carcinogen complex).
-what does this do to the DNA? It distorts the normal double helix and causes errors in the base sequence or mutations.
Benzo[a]pyrene is activated to have an epoxide group.
DNA-carcinogen complexes are called DNA adducts.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

12. Site of DNA Adduct Formation
As this slide shows there are numerous electron sites on a DNA molecule that could serve as a site for carcinogen attachment.
-Although chemical carcinogens fall into a variety of categories, many of them can be converted into electrophilic molecules that in turn become linked to DNA.
-This is important because the ability of a chemical to form DNA adducts is really the best gauge of its likelihood of causing cancer.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

13. Carcinogens and DNA Damage
In addtion to forming DNA adducts there are numeous other ways that carcingens can cause damage to DNA.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

14. Chemical Carcinogenesis
Chemical carcinogenesis is a multistep process.
Rous observed that repeated application of tar caused tumors in rabbits.
There are two main steps
Tumor initiation
Tumor Promotion
Peyton rous (1940s) first proposed that cancer was a multistep process.
-He observe that repeated application of coal tar to rabbit skin caused tumors to develop, but that the tumors would go away when the coal tar was removed.
-He also observed that when a skin irritant like turpentine was applied to the skin that the tumors reappeared. Turpentine does not cause tumors on its own.
-Collectively these expts suggested that coal tar and turpentine play two different roles in cancer development. He called these two roles tumor initiation and promotion.
-tumor initiation converts normal cells into a precancerous state and then promotion stimulates the precancerous cell to divide and form tumors.
-One of the components of coal tar is a chemical known as DMBA (dimethylbenz[a]antrhacene. It is a potent carcinogen,but giving mice a single dose of this rarely causes cancer.
-However if the skin of mice is given a single dose of DMBA followed by an irritant such as Croton oil (a plant oil), then cancer does develop in the treated area.
-It is possible for months or years to go by between the two treatments and cancer will still develop.
-this expt supports the idea that cancer is a multistep process involving both tumor initiators and promoters.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

15. Tumor Initiation
Since years may exist between treating animals with DMBA and croton oil with the same outcome. It seems that the single DMBA treatment must be able to permanently alter the DNA, creating a permanmently altered inititated state throughout the body. The tumor promoter (croton oil) can then act on the altered cells to promote tumor development.
-This permanent alteration to the body is typically in the form of a DNA mutation. So carcinogens that are capable of inducing a permanent somatic mutation is referred to as genotoxic because they cause damage to the genes
-What about DNA repair mechanisms? They still have the chance to repair the DNA, but they must work quickly. Once the cell undergoes DNA replication in preparation for cell division, the mutation becomes nearly impossible to remove.
-In this figure, the carcinogen methynitrosourea attacks the base Guanine in DNA creating a methylated derivative. If the cell cannot repair the damage before replication takes place then, the methylated guanine will pair with thymine duing replication (instead of cytosine). This creates a permanent mutation in which a G was converted to a T.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

16. Tumor Promotion
Like we just discussed tumor initiation is a single event. In contrast the process of tumor promotions is gradual and relies on prolonged or repeated exposures to a promoting agent.
-It has the ability to stimulate proliferation and if removed a promoter early in the process may eliminate tumor formation.
-Why is it that tumor may go away if the promoter is removed? Early in tumor promotion, the promoter is stimulating proliferation of intiated cells which creates more initiated (damaged cells). Early in the process the cell proliferation is dependent on the presence of the tumor promoter, so if it is removed the tumor cells will stop growing. However as cell division continues, natural selection will favor the cells whose proliferation is faster and autonomous. This will eventually lead to the development of a malignant tumor….whose growth no longer depends on the tumor promoter.
-Phorbol esters serve as the first established mechanism of how promoting agents serve to stimulate cell proliferation. They are the class tumor promoters found in croton oil. The most potent phorbol ester is tetradecanoyl phorbol acetate (TPA). TPA binds to and activates protein kinase C (a component of a signaling pathway the stimulates cell proliferation). Normally external signaling molecules bind to the surface receptors whose activation leads to the production of diacylglycerol (DAG). The DAG then activates protein kinase C, which triggers events leading to cell division. Phorbol esters mimic the action of DAG and serve to bind to and activate protein kinase C directly.
-However, unlike DAG which is eventually converted to an inactive form….phorbol esters continually activate protein kinase C.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

17. TPA and Tumor Promotion
The activation of protein kinase C by TPA is a very selective interaction. Tiny changes in the chemical structure of TPA generates derivatives that have a diminished ability to bind to protein kinase C. Therefore, they have a substantially reduced tumor promoting activity as shown here.
-the side chain alterations present in these two derivatives show a dramatic decrease in the ability of the molecule to bind to protein kinase C.
-TPA is just one example, there are numerous substances that stimulate cells proliferation and act as tumor promoting agents. Examples of this include estrogen, testosterone, asbestos, the fungal toxin teleocidin and alcohol.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

18. Main Stages of Carcinogenesis
We have mentioned over and over that cancer is a multistep process…..part of this stems from multiple tumor promoting steps and part form the repeated cycles of selection for rapid growth or other survival promoting properties.
-Rous’ original model of carcinogenesis only involved two steps…..initiation and promotion. However it is now clear that there is a 3rd step of tumor progression which follows tumor promotion.
-Tumor progression involves the multiple gradual changes in the properties of a proliferating cell that occur over time as cells acquire more aggressive traits of proliferation and invasion.
-Essentially this is a process of selecting the cells with the greatest proliferative and invasive qualities…..such as increased growth rate, increased invasiveness, abiility to survive in the bloodstream, resistance to immune attack and ability to grow in other organs.
-It is easy to understand why the highly aggressive and proliferative cells have the growth advantage, but how do these cells acquire the ability to do this”
-acquisition of new mutations…..if the new mutation causes the cells to divide more rapidly, then they will most likely outgrow their companion cells and become the primary cell type in the tumor.
-remember we talked about this is chapter 2…..it is called clonal selection. The predominant cells are called clonal.
-multiple cycles of mutation and selection can occur in succession…with each new population having enhanced growth over the last.
-in additon to mutations, cancer cell properties can be affected by changes in the expression of normal genes….these are referred to as epigenetic changes (any change in gene expression that does not involve a mutation). The traits required for cancer cell invasion and metastasis are controlled by epigenetic changes.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

19. Transplantation of Cancer Nuclei
Mutations are difficult to reverse, but epigenetic changes are more easy to reverse…..so is it possible to reverse epigenetic changes responsible for malignant changes to cells.
-This can be addressed experimentally by transferring the nucleus of a cancer cell into a different cytoplasmic environment to see it its gene expression patterns can be reverted to their normal state. When cancer nuclei are transplanted into mouse egg cells with their nuclei removed, the eggs divide and go through the early embyonic development despite the presence of the cancer cell nuclei.
-when mouse melanoma cells are used for this experiment, the eggs receiving the melanoma nuclei divide and produce embryonic cells that give rise to normal appearing cells and tissues of adult mice. However, these mice do still have an increased propensity for developing cancer.
-This expt shows that both epigenetic and genetic factors play a role in tumor development.
In summary:
What is important to tumor progression: gradual acquisition of DNA muations, epigenetic changes, and the natural selection to generate clonal population of cells
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

20. Carcinogenic Potential
Incomplete Carcinogens
Complete Carcinogens
Cancer Potency is influenced by
Activation reactions
Electrophillic stength of carcinogens
Some carcinogens only act as promoters or initiators while other act as both.
-In some cases the ability of a chemical to act as a complete carcinogen may be dependent dose. There is a certain dose needed to for the carcinogen act as both a promoter and initiator.
-Cancer causes may be complicated…..incomplete carcinogens do not cause cancer on their own, but they do contribute to the development of cancer. In general exposure to chemical carcinogens increases the propensity of cancer development.
-Another reason that dose is important comes from the concept that increased dose of an initiator will lead to accumulation of more DNA adducts and other types of DNA damage. However, to initiate cancer this DNA damage must occur in specific genes (we will address these in ch 9 and 10). Carcinogens introduce mutation randomly….so it is not probably that a random mutation will occur in a cancer specific gene. However, the more damage that is induced by a high dose of a carcinogen or continued exposure to a carcinogen, the greater the chance that one of these cancer specific genes may be affected.
Cancer potency – in animals this is measured by the amount of a chemical need to cause cancer in 50% of the animals tested. These tests have demonstrated a 10 million fold difference in strength separates the strongest carcinogens from the weakest carcinogens.
-What is responsible to mediate the variation in strength of carcinogens?
1. Activation reaction catalyzed by cytochrome p450
2. The electrophillic nature of substance….some are highly electrophillic and more reactive with DNA than others.
From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings.

21. Random Nature of Cancer
Potency and Dose of carcinogens
Random occurrence of mutations
Genetic risk factors contribute to acquisition and progression of cancer.
Immunosuppressive drugs contribute to cancer development.