Chapter 2 – Part II Chemical- and Radiation-induced Carcinogenesis Mar 8 & 13, 2007

Causes of cancer?

Causes of cancer Physical → radiation Chemical → chemicals Biological → microorganisms, especially viruses

2.8 Specific chemical agents can induce cancer [historical perspectives] The evidence that chemicals can induce cancer in humans has been accumulating for more than two centuries. Hill (1761) – Nasal cancers occurred in people who used snuff excessively. Pott (1775) – a high incidence of scrotal ( 陰囊 ) skin cancer among men who had spent their childhood as chimney sweeps

von Volkman & Bell (~ 1875) – Skin cancers occurred in workers whose skin was in continuous contact with tar ( 焦油 ) and paraffin oils ( 石蠟油 ), (now known as polycyclic aromatic hydrocarbons, PAH) Rehn (1895) – development of urinary bladder cancer in aniline ( 苯胺 ) dye workers

Similar observations were later made in many laboratories and established a relationship between heavy exposure to 2-naphthylamine, benzidine, or 4-amino-biphenyl and bladder cancer. Yamagiwa & Ichikawa (1915) induced skin carcinomas by the repeated application of coal tar to the ears of rabbits. – the 1st animal experiment demonstrating chemical carcinogenesis. Yamagiwa 山極

Direct evidence for a carcinogenic effect of the polycyclic aromatic hydrocarbons in tars (1930s): Kennaway & Hieger – synthetic 1,2,5,6- dibenzanthracene was a carcinogen. Cook, Hewitt & Hieger – identification of the carcinogen 3,4- benzpyrene in coal tar Since 1940s, the list of known carcinogenic chemicals has been expanded tremendously.

Carcinogenic hydrocarbons PAH

Chemical carcinogens: chemicals that cause tumor formation 1. have a very broad range of structures with no obvious unifying features 2. are genotoxic and can be classified into two broad categories based on their action mechanisms: a. Direct-acting carcinogens - react with nitrogen and/or oxygen atoms in DNA example: ethylmethane sulfonate (EMS) b. Indirect-acting carcinogens - become reactive after metabolic activation examples: aflatoxin, benzo[a]pyrene * genotoxic: an agent or process that interacts with cellular DNA, resulting in alteration of DNA structure

The direct-acting carcinogens interact with macromolecules through the covalent bond formation between an electrophilic form of the carcinogen and the nucleophilic sites in proteins (e.g., S, O, and N atoms in cysteine, tyrosine, and histidine, respectively) and nucleic acids (e.g., N and O atoms in purine or pyrimidine), such as N-methyl-N-nitrosourea, a chemically-reactive alkylating agent. Some agents can intercalate ( 嵌入 ) into the DNA double helix by forming tight noncovalent bonds (e.g., daunorubicin).

Most of carcinogens are indirectly-acting; they do not interact in vitro with macro- molecules until it has been incubated with liver homogenates or liver microsomal fractions. Thus, metabolic activation of certain carcinogenic agents is necessary to produce the “ultimate carcinogen” that actually reacts with crucial molecules in target cells.

1. Cytochrome P450 catalyses initial epoxidation. 2. With the exception of the and oxides that convert to phenols, epoxide hydrolase may catalyze the formation of dihydrodiols. 3. Benzo[a]pyrene-7, 8-dihydrodiol is further metabolized at the olefinic double bond by cytochrome P450 to form a vicinal diol-epoxide (r7, t8-dihydroxy-c9, 10 epoxy-7,8,9,10- tetrahydroxybenzo[a]pyrene). 4. The highly unstable arene ring opens spontaneously to form a carbocation. 5. This electrophilic species forms a covalent bond between the 10 position of the hydrocarbon and the exocyclic amino group of deoxyguanosine. Metabolic activation of benzo[a]pyrene electrophilic DNA adducts guanine

Metabolic activation of aflatoxin epoxide Aflatoxin B1, a toxin from a mold (Aspergillus flavus oryzae) that grows on grain and peanuts when they are stored under humid tropical conditions. It is thought to be a contributory cause of liver cancer in the tropics. (procarcinogen) (ultimate carcinogen)

DNA adduct formation Since most chemical carcinogens react with DNA and are mutagenic, interactions with DNA have been viewed as the most important reactions of these agents. The principal reaction products of the nitrosamines and similar alkylating agents with DNA are N 7 and O 6 guanine derivatives. Reactions also occur with other DNA bases.

A. N-7 (benzo[a]pyren-6-yl)guanine B. N-(deoxyguanosin-8-yl)-{acetyl}aminobiphenyl C. 8,9-dihydro-8-(N5-formyl- 2, 5, 6 -triamino-4 -oxo-N5-pyrimidyl)-9-hydroxy-aflatoxin B1 D. O 6 -[4-Oxo-4(3-pyridyl)butyl]guanine, a mutagenic lesion formed by the metabolism of the tobacco-specific nitrosamine, NNK E. N-7-methyldeoxyguanosine Examples of carcinogen- DNA adducts O6O6 N7N7 deoxyguanosine

a. An insertion of the flat planar rings of a polycyclic hydrocarbon between the stacked bases of double-helical DNA may distort the helix, leading to a frame- shift mutation during DNA replication past the point of the intercalation. Potential biological consequences of DNA-adduct formation

b. Alkylated bases in DNA can mispair with the wrong base during DNA replication – for example, O 6 methyguanine pairs with thymine instead of cytosine during DNA replication, leading to a base transition (i.e., GC→AT) type of mutation during the next round of DNA replication.

c. Many of the base adducts formed by carcinogens involve modifications of N-3 or N-7 positions on purines that induce an instability in the glycosidic bond between the purine base and deoxyribose. This destabilized structure can then undergo cleavage by DNA glycosylase, resulting in loss of the base.

Purines and Pyrimidines

d. Interaction with some carcinogens has been shown to favor a conformational transition of DNA from its usual double- helical B form to a Z-DNA form. This could alter the transcribability of certain genes, since B → Z conformational transitions are thought to be involved in regulating chromatin structure.

Radiation-induced Carcinogenesis Radiations contain energies greater than that in chemical bonds. Therefore, chemical bonds can be broken by radiation. Energy release from various radiations: Atomic particles > X-rays > ultraviolet (UV) light > visible light

Röntgen discovered x-rays in The harmful effects of x-rays were observed soon after their discovery. The first observed effects were the acute ones, such as reddening and blistering of the skin within hours or days after exposure. By 1902, it became apparent that cancer was one of the possible delayed effects of x-ray exposure. *Wilhelm Röntgen (1845 – 1923), a German physicist, received the Nobel Prize in physics in 1901.

2.9 Both physical and chemical carcinogens act as mutagens In 1927, Muller discovered that the phenotypes of Drosophila were changed by exposing the flies to X-rays. * Hermann Muller (1890 – 1967), an American geneticist, received the Nobel Prize in Medicine & Physiology in 1946.

The ability of radiation to cause human cancer, especially leukemia, was dramatically shown by the increased rates of leukemia among survivors of the atomic bombs dropped in World War II, and more recently by the increase in skin cancer in individuals exposed to too much sunlight (UV radiation).

UV radiation is a low-energy emission and does not penetrate deeply. Hence, the skin absorbs most of the radiation and is the primary carcinogenic target.

A number of the points made about chemical carcinogenesis are also true for radiation-induced carcinogenesis. Both x-rays and ultraviolet (UV) radiation cause DNA damage.

When cells are exposed to UV light in the 240- to 300-nm range, nucleic acid bases acquire excited energy states, producing photochemical reactions between DNA bases. The principal products in DNA at biologically relevant doses of UV light are cyclobutane dimers formed between two adjacent pyrimidine bases in the DNA chain. Both thymine- thymine and thymine-cytosine dimers are formed.

Cyclobutane dimer

Ames test Bruce Ames (1975) - measured the ability of carcinogens to mutate the bacteria

Figure 2.25 The Biology of Cancer (© Garland Science 2007) Chemicals that are potently mutagenic are also powerful carcinogens

Are all mutagens carcinogens? Are all carcinogens mutagens?