Mutagen

The international pictogram for chemicals that are sensitising, mutagenic, carcinogenic or toxic to reproduction.

In genetics, a mutagen is a physical or chemical agent that changes the genetic material, usually DNA, of an organism and thus increases the frequency of mutations above the natural background level. As many mutations can cause cancer, mutagens are therefore also likely to be carcinogens, although not always necessarily so. Some chemicals only become mutagenic through cellular processes. Not all mutations are caused by mutagens: so-called "spontaneous mutations" occur due to spontaneous hydrolysis, errors in DNA replication, repair and recombination.

Discovery of mutagens

The first mutagens to be identified were carcinogens, substances that were shown to be linked to cancer. Tumors were described more than 2,000 years before the discovery of chromosomes and DNA; in 500 B.C., the Greek physician Hippocrates named tumors resembling a crab karkinos (from which the word "cancer" is derived via Latin), meaning crab.^[1] In 1567, Swiss physician Paracelsus suggested that an unidentified substance in mined ore (identified as radon gas in modern times) caused a wasting disease in miners,^[2] and in England, in 1761, John Hill made the first direct link of cancer to chemical substances by noting that excessive use of snuff may cause nasal cancer.^[3] In 1775, Sir Percivall Pott wrote a paper on the high incidence of scrotal cancer in chimney sweeps, and suggested chimney soot as the cause of scrotal cancer.^[4] In 1915, Yamagawa and Ichikawa showed that repeated application of coal tar to rabbit's ears produced malignant cancer.^[5] Subsequently in the 1930s the carcinogen component in coal tar was identified as a polyaromatic hydrocarbon (PAH), benzo[a]pyrene.^[2][6] Polyaromatic hydrocarbons are also present in soot, which was suggested to be a causative agent of cancer over 150 years earlier.

The mutagenic property of mutagens was first demonstrated in 1927, when Hermann Muller discovered that x-rays can cause genetic mutations in fruit flies, producing phenotypic mutants as well as observable changes to the chromosomes,^[7] visible due to presence of enlarged 'polytene' chromosomes in fruit fly salivary glands.^[8] His collaborator Edgar Altenburg also demonstrated the mutational effect of UV radiation in 1928.^[9] Muller went on to use x-rays to create Drosophila mutants that he used in his studies of genetics.^[10] He also found that X-rays not only mutate genes in fruit flies but also have effects on the genetic makeup of humans.^[11] Similar work by Lewis Stadler also showed the mutational effect of X-ray on barley in 1928,^[12] and ultraviolet (UV) radiation on maize in 1936.^[13] The effect of sunlight had previously been noted in the nineteenth century where rural outdoor workers and sailors were found to be more prone to skin cancer.^[14]

Chemical mutagens were not demonstrated to cause mutation until the 1940s, when Charlotte Auerbach and J. M. Robson found that mustard gas can cause mutations in fruit flies.^[15] A large number of chemical mutagens have since been identified, especially after the development of the Ames test in the 1970s by Bruce Ames that screens for mutagens and allows for preliminary identification of carcinogens.^[16][17] Early studies by Ames showed around 90% of known carcinogens can be identified in Ames test as mutagenic (later studies however gave lower figures),^[18][19][20] and ~80% of the mutagens identified through Ames test may also be carcinogens.^[20][21] Mutagens are not necessarily carcinogens, and vice versa. Sodium azide for example may be mutagenic (and highly toxic), but it has not been shown to be carcinogenic.^[22]

Effects of mutagens

Mutagens can cause changes to the DNA and are therefore genotoxic. They can affect the transcription and replication of the DNA, which in severe cases can lead to cell death. The mutagen produces mutations in the DNA, and deleterious mutation can result in aberrant, impaired or loss of function for a particular gene, and accumulation of mutations may lead to cancer. Mutagens may therefore be also carcinogens. However, some mutagens exert their mutagenic effect through their metabolites, and therefore whether such mutagens actually become carcinogenic may be dependent on the metabolic processes of an organism, and a compound shown to be mutagenic in one organism may not necessarily be carcinogenic in another.^[23]

Different mutagens act on the DNA differently. Powerful mutagens may result in chromosomal instability,^[24] causing chromosomal breakages and rearrangement of the chromosomes such as translocation, deletion, and inversion. Such mutagens are called clastogens.

Mutagens may also modify the DNA sequence; the changes in nucleic acid sequences by mutations include substitution of nucleotide base-pairs and insertions and deletions of one or more nucleotides in DNA sequences. Although some of these mutations are lethal or cause serious disease, many have minor effects as they do not result in residue changes that have significant effect on the structure and function of the proteins. Many mutations are silent mutations, causing no visible effects at all, either because they occur in non-coding or non-functional sequences, or they do not change the amino-acid sequence due to the redundancy of codons.

Some mutagens can cause aneuploidy and change the number of chromosomes in the cell.

In Ames test, where the varying concentrations of the chemical are used in the test, the dose response curve obtained is nearly always linear, suggesting that there is no threshold for mutagenesis. Similar results are also obtained in studies with radiations, indicating that there may be no safe threshold for mutagens. However, some proposed that low level of some mutagens may stimulate the DNA repair processes and therefore may not necessarily be harmful.

Types of mutagens

Mutagens may be of physical, chemical or biological origin. They may act directly on the DNA, causing direct damage to the DNA, and most often result in replication error. Some however may act on the replication mechanism and chromosomal partition. Many mutagens are not mutagenic by themselves, but can form mutagenic metabolites through cellular processes, for example through the activity of the cytochrome P450 system and other oxygenases such as cyclooxygenase.^[25] Such mutagens are called promutagens.

Physical mutagens

• Ionizing radiations such as X-rays, gamma rays and alpha particles may cause DNA breakage and other damages. The most common sources include cobalt-60 and cesium-137.
• Ultraviolet radiations with wavelength above 260 nm are absorbed strongly by bases, producing pyrimidine dimers, which can cause error in replication if left uncorrected.
• Radioactive decay, such as ¹⁴C in DNA which decays into nitrogen.

DNA reactive chemicals

A DNA adduct (at center) of the mutagenic metabolite of benzo[a]pyrene from tobacco smoke.

A large number of chemicals may interact directly with DNA. However, many such as PAHs, aromatic amines, benzene are not necessarily mutagenic by themselves, but through metabolic processes in cells they produce mutagenic compounds.

• Reactive oxygen species (ROS) – These may be superoxide, hydroxyl radicals and hydrogen peroxide, and large number of these highly reactive species are generated by normal cellular processes, for example as a by-products of mitochondrial electron transport, or lipid peroxidation. As an example of the latter, 15-hydroperoxyicosatetraenocic acid, a natural product of cellular cyclooxygenases and lipoxygenases, breaks down to form 4-hydroxy-2(E)-nonenal, 4-hydroperoxy-2(E)-nonenal, 4-oxo-2(E)-nonenal, and cis-4,5-epoxy-2(E)-decanal; these bifunctional electophils are mutagenic in mammalian cells and may contribute to the development and/or progression of human cancers (see 15-Hydroxyicosatetraenoic acid).^[26] A number of mutagens may also generate these ROS. These ROS may result in the production of many base adducts, as well as DNA strand breaks and crosslinks.
• Deaminating agents, for example nitrous acid which can cause transition mutations by converting cytosine to uracil.
• Polycyclic aromatic hydrocarbon (PAH), when activated to diol-epoxides can bind to DNA and form adducts.
• Alkylating agents such as ethylnitrosourea. The compounds transfer methyl or ethyl group to bases or the backbone phosphate groups. Guanine when alkylated may be mispaired with thymine. Some may cause DNA crosslinking and breakages. Nitrosamines are an important group of mutagens found in tobacco, and may also be formed in smoked meats and fish via the interaction of amines in food with nitrites added as preservatives. Other alkylating agents include mustard gas and vinyl chloride.
• Aromatic amines and amides have been associated with carcinogenesis since 1895 when German physician Ludwig Rehn observed high incidence of bladder cancer among workers in German synthetic aromatic amine dye industry. 2-Acetylaminofluorene, originally used as a pesticide but may also be found in cooked meat, may cause cancer of the bladder, liver, ear, intestine, thyroid and breast.
• Alkaloid from plants, such as those from Vinca species, may be converted by metabolic processes into the active mutagen or carcinogen.
• Bromine and some compounds that contain bromine in their chemical structure.
• Sodium azide, an azide salt that is a common reagent in organic synthesis and a component in many car airbag systems
• Psoralen combined with ultraviolet radiation causes DNA cross-linking and hence chromosome breakage.
• Benzene, an industrial solvent and precursor in the production of drugs, plastics, synthetic rubber and dyes.

Base analogs

• Base analog, which can substitute for DNA bases during replication and cause transition mutations.

Intercalating agents

• Intercalating agents, such as ethidium bromide and proflavine, are molecules that may insert between bases in DNA, causing frameshift mutation during replication. Some such as daunorubicin may block transcription and replication, making them highly toxic to proliferating cells.

Metals

Many metals, such as arsenic, cadmium, chromium, nickel and their compounds may be mutagenic, but they may act, however, via a number of different mechanisms.^[27] Arsenic, chromium, iron, and nickel may be associated with the production of ROS, and some of these may also alter the fidelity of DNA replication. Nickel may also be linked to DNA hypermethylation and histone deacetylation, while some metals such as cobalt, arsenic, nickel and cadmium may also affect DNA repair processes such as DNA mismatch repair, and base and nucleotide excision repair.^[28]

Biological agents

• Transposon, a section of DNA that undergoes autonomous fragment relocation/multiplication. Its insertion into chromosomal DNA disrupt functional elements of the genes.
• Virus – Virus DNA may be inserted into the genome and disrupts genetic function. Infectious agents have been suggested to cause cancer as early as 1908 by Vilhelm Ellermann and Oluf Bang,^[29] and 1911 by Peyton Rous who discovered the Rous sarcoma virus.^[30]
• Bacteria – some bacteria such as Helicobacter pylori cause inflammation during which oxidative species are produced, causing DNA damage and reducing efficiency of DNA repair systems, thereby increasing mutation.

Protection against mutagens

Fruits and vegetables are rich in antioxidants.

Antioxidants are an important group of anticarcinogenic compounds that may help remove ROS or potentially harmful chemicals. These may be found naturally in fruits and vegetables.^[31] Examples of antioxidants are vitamin A and its carotenoid precursors, vitamin C, vitamin E, polyphenols, and various other compounds. β-Carotene is the red-orange colored compounds found in vegetables like carrots and tomatoes. Vitamin C may prevent some cancers by inhibiting the formation of mutagenic N-nitroso compounds (nitrosamine). Flavonoids, such as EGCG in green tea, have also been shown to be effective antioxidants and may have anti-cancer properties. Epidemiological studies indicate that a diet rich in fruits and vegetables is associated with lower incidence of some cancers and longer life expectancy,^[32] however, the effectiveness of antioxidant supplements in cancer prevention in general is still the subject of some debate.^[32][33]

Other chemicals may reduce mutagenesis or prevent cancer via other mechanisms, although for some the precise mechanism for their protective property may not be certain. Selenium, which is present as a micronutrient in vegetables, is a component of important antioxidant enzymes such as gluthathione peroxidase. Many phytonutrients may counter the effect of mutagens; for example, sulforaphane in vegetables such as broccoli has been shown to be protective against prostate cancer.^[34] Others that may be effective against cancer include indole-3-carbinol from cruciferous vegetables and resveratrol from red wine.^[35]

An effective precautionary measure an individual can undertake to protect themselves is by limiting exposure to mutagens such as UV radiations and tobacco smoke. In Australia, where people with pale skin are often exposed to strong sunlight, melanoma is the most common cancer diagnosed in people aged 15–44 years.^[36][37]

In 1981, human epidemiological analysis by Richard Doll and Richard Peto indicated that smoking caused 30% of cancers in the US.^[38] Diet is also thought to cause a significant number of cancer, and it has been estimated that around 32% of cancer deaths may be avoidable by modification to the diet.^[39] Mutagens identified in food include mycotoxins from food contaminated with fungal growths, such as aflatoxins which may be present in contaminated peanuts and corn; heterocyclic amines generated in meat when cooked at high temperature; PAHs in charred meat and smoked fish, as well as in oils, fats, bread, and cereal;^[40] and nitrosamines generated from nitrites used as food preservatives in cured meat such as bacon (ascobate, which is added to cured meat, however, reduces nitrosamine formation).^[31] Excessive alcohol consumption has also been linked to cancer; the possible mechanisms for its carcinogenicity include formation of the possible mutagen acetaldehyde, and the induction of the cytochrome P450 system which is known to produce mutagenic compounds from promutagens.^[41]

For certain mutagens, such as dangerous chemicals and radioactive materials, as well as infectious agents known to cause cancer, government legislations and regulatory bodies are necessary for their control. ^[42]

Mutagen test systems

Many different systems for detecting mutagen have been developed.^[43][44] Animal systems may more accurately reflect the metabolism of human, however, they are expensive and time-consuming (may take around three years to complete), they are therefore not used as a first screen for mutagenicity or carcinogenicity.

Bacterial systems

• Ames test – This is the most commonly used test, and Salmonella typhimurium strains deficient in histidine biosynthesis are used in this test. The test checks for mutants that can revert to wild-type. It is an easy, inexpensive and convenient initial screen for mutagens.
• Resistance to 8-azaguanine in S. typhimurium – Similar to Ames test, but instead of reverse mutation, it checks for forward mutation that confer resistance to 8-Azaguanine in a histidine revertant strain.
• Escherichia coli systems – Both forward and reverse mutation detection system have been modified for use in E. coli. Tryptophan-deficient mutant is used for the reverse mutation, while galactose utility or resistance to 5-methyltryptophan may be used for forward mutation.
• DNA repair – E. coli and Bacillus subtilis strains deficient in DNA repair may be used to detect mutagens by their effect on the growth of these cells through DNA damage.

Yeast

Systems similar to Ames test have been developed in yeast. Saccharomyces cerevisiae is generally used. These systems can check for forward and reverse mutations, as well as recombinant events.

Drosophila

Sex-Linked Recessive Lethal Test – Males from a strain with yellow bodies are used in this test. The gene for the yellow body lies on the X-chromosome. The fruit flies are fed on a diet of test chemical, and progenies are separated by sex. The surviving males are crossed with the females of the same generation, and if no males with yellow bodies are detected in the second generation, it would indicate a lethal mutation on the X-chromosome has occurred.

Plant Assays

Plants such as Zea mays, Arabidopsis thaliana and Tradescantia have been used in various test assays for mutagenecity of chemicals.

Cell culture assay

Mammalian cell lines such as Chinese hamster V79 cells, Chinese hamster ovary (CHO) cells or mouse lymphoma cells may be used to test for mutagenesis. Such systems include the HPRT assay for resistance to 8-azaguanine or 6-thioguanine, and ouabain-resistance (OUA) assay.

Rat primary hepatocytes may also be used to measure DNA repair following DNA damage. Mutagens may stimulate unscheduled DNA synthesis that results in more stained nuclear material in cells following exposure to mutagens.

Chromosome check systems

These systems check for large scale changes to the chromosomes and may be used with cell culture or in animal test. The chromosomes are stained and observed for any changes. Sister chromatid exchange is a symmetrical exchange of chromosome material between sister chromatids and may be correlated to the mutagenic or carcinogenic potential of a chemical. In micronucleus Test, cells are examined for micronuclei, which are fragments or chromosomes left behind at anaphase, and is therefore a test for clastogenic agents that cause chromosome breakages. Other tests may check for various chromosomal aberrations such as chromatid and chromosomal gaps and deletions, translocations, and ploidy.

Animal test systems

Rodents are usually used in animal test. The chemicals under test are usually administered in the food and in the drinking water, but sometimes by dermal application, by gavage, or by inhalation, and carried out over the major part of the life span for rodents. In tests that check for carcinogens, maximum tolerated dosage is first determined, then a range of doses are given to around 50 animals throughout the notional lifespan of the animal of two years. After death the animals are examined for sign of tumours. Differences in metabolism between rat and human however means that human may not respond in exactly the same way to mutagen, and dosages that produce tumours on the animal test may also be unreasonably high for a human, i.e. the equivalent amount required to produce tumours in human may far exceed what a person might encounter in real life.

Mice with recessive mutations for a visible phenotype may also be used to check for mutagens. Females with recessive mutation crossed with wild-type males would yield the same phenotype as the wild-type, and any observable change to the phenotype would indicate that a mutation induced by the mutagen has occurred.

Mice may also be used for dominant lethal assays where early embryonic deaths are monitored. Male mice are treated with chemicals under test, mated with females, and the females are then sacrificed before parturition and early fetal deaths are counted in the uterine horns.

Transgenic Mouse Assay using a mouse strain infected with a viral shuttle vector is another method for testing mutagens. Animals are first treated with suspected mutagen, the mouse DNA is then isolated and the phage segment recovered and used to infect E. coli. Using similar method as the blue-white screen, the plaque formed with DNA containing mutation are white, while those without are blue.

Use of mutagen in anti-cancer therapy

Many mutagens are highly toxic to proliferating cells, and they are often used to destroy cancer cells. Alkylating agents such as cyclophosphamide and cisplatin, as well as intercalating agent such as daunorubicin and doxorubicin may be used in chemotherapy. However, due to its effect on other cells which are also rapidly dividing, they may have side effects such as hair loss and nausea. Research on better targeted therapies may reduce such side-effects. Ionizing radiations are used in radiation therapy.

Mutagens in fiction

In science fiction, mutagens are often represented as substances that are capable of completely changing the form of the recipient or gaining them superpower. Powerful radiations are the agents of mutation for the superheroes in Marvel Comics's Fantastic Four, Daredevil, and Hulk, while in the Teenage Mutant Ninja Turtles franchise the mutagen is chemical agent called the Ooze, and for Inhumans the mutagen is the Terrigen Mist. Mutagens are also featured in television series, computer and video games, such as the Cyberia, The Witcher, Metroid Prime: Trilogy, Resistance: Fall of Man, Resident Evil, Infamous, Command & Conquer, Gears of War 3, and Fallout.

See also

• Antimutagen
• Carcinogen
• DNA repair
• Ethyl methanesulfonate (EMS)
• Genetics
• Genotoxicity
• Mutation
• Pesticide
• Teratology

References

1. Niki Papavramidou; Theodossis Papavramidis; Thespis Demetriou (2010). "Ancient Greek and Greco-Roman Methods in Modern Surgical Treatment of Cancer". Annals of Surgical Oncology. 17 (3): 665–7. doi:10.1245/s10434-009-0886-6. PMC 2820670. PMID 20049643.
2. Nature and Nurture – Lessons from Chemical Carcinogenesis: Chemical Carcinogens – From Past to Present
3. Hill, J. Cautions Against the Immoderate Use of Snuff. Founded on the Known Qualities of the Tobacco Plant; And the Effects it Must Produce when this Way Taken into the Body: And Enforced by Instances of Persons who have Perished Miserably of Diseases, Occasioned, or Rendered Incurable by its Use (R. Baldwin and J. Jackso, London, 1761).
4. Brown, J. R.; Thornton, J. L. (1957). "Percivall Pott (1714-1788) and Chimney Sweepers' Cancer of the Scrotum". British journal of industrial medicine. 14 (1): 68–70. doi:10.1136/oem.14.1.68. PMC 1037746. PMID 13396156.
5. Yamagawa K, Ichikawa K (1915). "Experimentelle Studie ueber die Pathogenese der Epithel geschwuelste". Mitteilungen aus der medizinischen Fakultät der Kaiserlichen Universität zu Tokyo. 15: 295–344.
6. Cook, J. W., Hewett, C. L. & Hieger, I. (1933). "The isolation of a cancer-producing hydrocarbon from coal tar". Journal of Chemical Society. 24: 395–405. doi:10.1039/JR9330000395.
7. Muller, H. J. (1927). "Artificial Transmutation of the Gene" (PDF). Science. 66 (1699): 84–87. doi:10.1126/science.66.1699.84. PMID 17802387.
8. Griffiths, Anthony J. F.; Wessler, Susan R.; Carroll, Sean B.; Doebley, John (2012). Introduction to Genetic Analysis (10th ed.). W. H. Freeman. p. 255. ISBN 978-1-4292-7634-4.
9. The limit of radiation frequency effective in producing mutations Altenburg, E. (1928) The American Naturalist, 62, 540–545.
10. Crow, J. F.; Abrahamson, S. (1997). "Seventy Years Ago: Mutation Becomes Experimental". Genetics. 147 (4): 1491–1496. PMC 1208325. PMID 9409815.
11. Campbell, Neil A. and Jane B. Reece. (2005). Biology (7th ed.). San Francisco, CA: Pearson Education, Inc,. ISBN 080537146X.
12. Stadler, L. J. (1928). "Mutations in Barley Induced by X-Rays and Radium". Science. 68 (1756): 186–187. doi:10.1126/science.68.1756.186. PMID 17774921.
13. Stadler, L. J.; G. F. Sprague (1936-10-15). "Genetic Effects of Ultra-Violet Radiation in Maize. I. Unfiltered Radiation" (PDF). Proceedings of the National Academy of Sciences of the United States of America. US Department of Agriculture and Missouri Agricultural Experiment Station. 22 (10): 572–578. Bibcode:1936PNAS...22..572S. doi:10.1073/pnas.22.10.572. PMC 1076819. PMID 16588111. Retrieved 2007-10-11.
14. Hockberger, P. E. (2002). "A history of ultraviolet photobiology for humans, animals and microorganisms". Photochem. Photobiol. 76 (6): 561–579. doi:10.1562/0031-8655(2002)0760561AHOUPF2.0.CO2. PMID 12511035.
15. Charlotte Auerbach, J. M. Robson, & J. G. Carr (Mar 1947). "Chemical Production of Mutations". Science. 105 (2723): 243–247. Bibcode:1947Sci...105..243A. doi:10.1126/science.105.2723.243.
16. Ames, B. N.; Lee, F. D.; Durston, W. E. (1973). "An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens". Proceedings of the National Academy of Sciences of the United States of America. 70 (3): 782–786. doi:10.1073/pnas.70.3.782. PMC 433358. PMID 4577135.
17. Ames, B. N. (1979). "Identifying environmental chemicals causing mutations and cancer" (PDF). Science. 204 (4393): 587–593. doi:10.1126/science.373122. JSTOR 1748159. PMID 373122.
18. McCann, J.; Choi, E.; Yamasaki, E.; Ames, B. N. (1975). "Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals". Proceedings of the National Academy of Sciences of the United States of America. 72 (12): 5135–5139. doi:10.1073/pnas.72.12.5135. PMC 388891. PMID 1061098.
19. McCann J, Gold LS, Horn L, McGill R, Graedel TE, Kaldor J (1988). "Statistical analysis of Salmonella test data and comparison to results of animal cancer tests" (PDF). Mutation Research. 205 (1–4): 183–195. doi:10.1016/0165-1218(88)90017-1. PMID 3285186.
20. Dunkel VC, Zeiger E, Brusick D, McCoy E, McGregor D, Mortelmans K, Rosenkranz HS, Simmon VF (1985). "Reproducibility of microbial mutagenicity assays: II. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli". Environmental Mutagenenesis. 7 (suppl. 5): 1–248. PMID 3905369.
21. Romualdo Benigni; Cecilia Bossa (2011). "Alternative strategies for carcinogenicity assessment: an efficient and simplified approach based on in vitro mutagenicity and cell transformation assays" (PDF). Mutagenesis. 26 (3): 455–460. doi:10.1093/mutage/ger004. PMID 21398403.
22. Toxicology And Carcinogenesis Studies Of Sodium Azide
23. J.W. Allen, G.K. DeWeese, J.B. Gibson, P.A. Poorman, M.J. Moses (2003). "Synaptonemal complex damage as a measure of chemical mutagen effects on mammalian germ cells". 190 (1): 19–24. doi:10.1016/0165-7992(87)90076-5.
24. Huang, L.; Snyder, A. R.; Morgan, W. F. (2003). "Radiation-induced genomic instability and its implications for radiation carcinogenesis". Oncogene. 22 (37): 5848–5854. doi:10.1038/sj.onc.1206697. PMID 12947391.
25. Kim D, Guengerich FP. (2005). "Cytochrome P450 activation of arylamines and heterocyclic amines". Annu Rev Pharmacol Toxicol. 45: 27–49. doi:10.1146/annurev.pharmtox.45.120403.100010. PMID 15822170.
26. J. Biol. Chem. 280:28337-28346, 2005
27. Valko, M.; Morris, H.; Cronin, M. T. (2005). "Metals, toxicity and oxidative stress" (PDF). Current medicinal chemistry. 12 (10): 1161–1208. doi:10.2174/0929867053764635. PMID 15892631.
28. "Health Risk Assessment Guidance for Metals – Mutagenicity" (PDF). EBRC.
29. Ellermann V.; Bang O. (1908). "Experimentelle Leukämie bei Hühnern". Zentralbl. Bakteriol. Parasitenkd. Infectionskr. Hyg. Abt. Orig. 46: 595–609.
30. Peyton Rous (1911). "A sarcoma of the fowl transmissible by an agent separable from the tumor cells". Journal of Experimental Medicine. 13 (4): 397–411. doi:10.1084/jem.13.4.397. PMC 2124874. PMID 19867421.
31. Carcinogens and Anticarcinogens in the Human Diet. National Academy Press. 1996. ISBN 0-309-05391-9.
32. Dolara P, Bigagli E, Collins A (2012). "Antioxidant vitamins and mineral supplementation, life span expansion and cancer incidence: a critical commentary". Eur J Nutr. 51 (7): 769–81. doi:10.1007/s00394-012-0389-2. PMID 22684632.
33. Li K, Kaaks R, Linseisen J, Rohrmann S (2012). "Vitamin/mineral supplementation and cancer, cardiovascular, and all-cause mortality in a German prospective cohort". Eur J Nutr. 51 (4): 407–13. doi:10.1007/s00394-011-0224-1. PMID 21779961.
34. Gibbs A, Schwartzman J, Deng V, Alumkal J (2009). "Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6". Proc Natl Acad Sci U S A. 106 (39): 16663–8. doi:10.1073/pnas.0908908106. PMC 2757849. PMID 19805354.
35. Gullett NP, Ruhul Amin AR, Bayraktar S, Pezzuto JM, Shin DM, Khuri FR, Aggarwal BB, Surh YJ, Kucuk O (2010). "Cancer prevention with natural compounds". Seminars in Oncology. 37 (3): 258–81. doi:10.1053/j.seminoncol.2010.06.014. PMID 20709209.
36. "Skin Cancer Facts and Figures". Retrieved 2010-07-02.
37. Skin-tone gene could predict cancer risk
38. Doll, R.; Peto, R. (1981). "The causes of cancer: Quantitative estimates of avoidable risks of cancer in the United States today". Journal of the National Cancer Institute. 66 (6): 1191–1308. doi:10.1093/jnci/66.6.1192. PMID 7017215.
39. Willett WC (1995). "Diet, nutrition, and avoidable cancer". Environ Health Perspect. 103 (Suppl 8): 165–70. doi:10.1289/ehp.95103s8165. PMC 1518978. PMID 8741778.
40. Scientific Committee on Food (4 December 2002). "Polycyclic Aromatic Hydrocarbons – Occurrence in foods, dietary exposure and health effects" (PDF). European Commission. Retrieved 21 August 2010.
41. Pöschl G, Seitz HK (2004). "Alcohol and cancer". Alcohol and Alcoholism. 39 (3): 155–65. doi:10.1093/alcalc/agh057. PMID 15082451.
42. Genetics and the Law II. Springer US. 1980. ISBN 978-1-4613-3080-6.
43. Hodgson, Ernest (2004). "Chapter 21". A Textbook of Modern Toxicology (3rd ed.). John Wiley & Sons. ISBN 0-471-26508-X.
44. Williams, Phillip L.; James, Robert C.; Roberts, Stephen M. (2000). Principles of Toxicology – Environmental and Industrial Applications (2nd ed.). John Wiley & Sons. ISBN 0-471-29321-0.