Mutagen

Resources

A mutagen is a compound that causes a mutation.

In the living cell, deoxyribonucleic acid (DNA) undergoes frequent chemical change, especially when it is being replicated. Most of these changes are quickly repaired. Those that are not, result in a mutation. Thus, mutation is a failure of DNA repair. Mutagens are chemicals or physical factors (such as radiation) that increase the rate of mutation in the cells of bacteria, plants, and animals (including humans). Most mutagens are of natural origin and are not just a modern phenomenon.

Very small doses of a mutagen usually have little effect while large doses of a mutagen could be lethal. DNA in the nuclei of all cells encodes proteins, which play important structural and functional (metabolic) roles in the cell. Mutagens typically disrupt the DNA of cells, causing changes in the proteins that the cell produces, which can lead to abnormally fast growth (cancer), or even cell death. In rare incidences, mutagens can even cause protein changes that are beneficial to the cell.

The first mutagens to be identified were carcinogens, or cancer-causing substances. Early physicians detected tumors in patients more than 2,000 years before the discovery of chromosomes and DNA. In 500 BC the Greek Hippocrates named crab-shaped tumors cancer (meaning crab).

In England in 1775, Dr. Percivall Pott wrote a paper on the high incidence of scrotal cancer in chimney sweeps who were typically boys small enough to fit inside chimneys and clean out the soot. Pott suggested that chimney soot contained carcinogens that could cause the growth of the warts seen in scrotal cancer. Over a 150 years later, chimney soot was found to contain hydrocarbons capable of mutating DNA.

In France in the 1890s, Bordeaux wine workers showed an unusually high incidence of skin cancer on the back of the neck. These workers spend their days bending over in the fields picking grapes, exposing the back of their necks to the sun. The ultraviolet (UV) radiation in natural sunlight was later identified as a mutagen.

Mutagens can be found in foods, beverages, and drugs. Sometimes a substance is mutagenic because it is converted in the body into something harmful. Regulatory agencies are responsible for testing food and drugs to insure that the public is not unknowingly exposed to mutagens. However, some mutagen-containing substances are not tightly controlled. One such substance is found in the tobacco of cigars and cigarettes.

Some mutagens occur naturally, and some are synthetic. Cosmic rays from space are natural, but they are mutagenic. Some naturally occurring viruses are considered mutagenic since they can insert themselves into host DNA. Hydrogen and atomic bombs are humanmade, and they emit harmful radiation. Radiation from nuclear bombs and gaseous particles from nitrogen mustard and acridine orange have been used destructively in war. On the other hand, some mutagens are used constructively to kill bacteria that could grow in human foods, such as the small doses of nitrites used to preserve some meat. Even though nitrites can be mutagenic, without the nitrites these meats could cause botulism; hence, the benefit outweighs the risk involved.

Mutagens affect DNA in different ways. Some mutagens, such as nitrogen mustard, bind to a base and cause it to make a different amino acid. These mutagens cause point mutations, because they change the genetic code at one point, so changing a protein’s amino acid sequence.

Mutagens such as acridine orange work by deleting or inserting one or more bases into the DNA molecule, shifting the frame of the triplet code for an amino acid. Deletion and insertion mutations causing “frame-shift” mutations can change a long string of amino acids, which can severely alter the structure and function of a protein product.

Normal cells recognize cues from their environment and respond with specific reactions, but cells impaired by a mutation do not behave or appear normal, and are said to be transformed.

The significance of mutations is profoundly influenced by the distinction between germline and soma. Mutations in somatic (body) cells are not transferred to offspring. Mutations that occur in a somatic cell, in the bone marrow or liver for example, may damage the cell, make the cell cancerous or even kill the cell. Whatever the effect, the ultimate fate of that somatic mutation is to disappear when the cell in which it occurred, or its owner, dies. However, mutated DNA can only be passed to the next generation if it is present in a germ cell such as spermatozoa and ova (eggs), each of which contribute half of the DNA of the new organism. Germline mutations will be found in every cell descended from the zygote to which that mutant gamete contributed. If an adult is successfully produced, every one of its cells will contain the mutation. Included among these will be the next generation of gametes, so if the owner is able to become a parent, that mutation will pass down to yet another generation.

Chemical mutagens are classified as alkylating agents, cross-linking agents, and polycyclic aromatic hydrocarbons (PAHs). Alkylating agents act by adding molecular components to DNA bases, which alters the protein product. Cross-linking agents create covalent bonds with DNA bases, while PAHs are metabolized by the human body into other potentially mutagenic molecules.

Radiation is another potent mutagen. For biologists, the most significant forms of radiation are light, heat, and ionizing radiation. Ionizing radiation can penetrate cells and create ions in the cell contents. These, in turn, can cause permanent alterations in DNA; that is, mutations. Ionizing radiation includes: x rays, gamma rays, and the subatomic particles—neutrons, electrons (“beta” particles), and alpha particles (helium nuclei). Ionizing radiation alters the way two strands of DNA interact. This high energy radiation passes through cells and tissues, cutting up any DNA in its path. It can rearrange entire sections of the chromosomes, altering relatively long stretches of DNA. UV radiation causes covalent bonds to form between neighboring thymine bases in the DNA, so altering the DNA product at that location.

Mutagens are often associated with specific cancers in humans. Aromatic amines are mutagens that can cause bladder cancer. Tobacco taken in the form of snuff contains mutagens that can cause nose tumors. Tobacco smoke contains mutagens such as PAHs and nitrosamine (a type of alkylating agent), as well as toxins such as carbon monoxide, cyanide, ammonia, arsenic, and radioactive polonium. Although tobacco products are legal and widely available, many physicians and government agencies warn about the health risks linking smoking with several types of cancer and heart disease.

In 1973, Bruce Ames introduced the most widely-used test to identify potential mutagens. Suspected mutagens are mixed with a defective strain of the bacteria Salmonella, which only grows if it is mutated. Substances that allow the Salmonella to grow are considered mutagenic.

In addition to mutagen-induced DNA changes, spontaneous mutations occur in the dividing cells of the human body every day. The nuclei of the cells have repair enzymes, which remove mutations and restore mutated DNA to its original form. If these natural DNA repair mechanisms fail to keep up with the rate of mutation or the repair mechanisms themselves are defective, disease can result. This latter case is seen in lung cancer due to cigarette smoking, where the nicotine in the smoke is thought to block an important repair process in the lungs.

KEY TERMS

Amino acid —An organic compound whose molecules contain both an amino group (-NH₂) and a carboxyl group (-COOH). One of the building blocks of a protein.

DNA —Deoxyribonucleic acid; the genetic material in a cell.

Frame shift mutation —Mutation caused by the deletion or insertion one or more bases into the DNA molecule, shifting the frame of the triplet code for an amino acid. Such mutations can change a long string of amino acids, thereby severely altering the structure and function of a protein product.

Genetic code —The blueprint for all structures and functions in a cell as encoded in DNA.

Mutation —A change made to DNA.

Point mutation —A change in the genetic code at one point, which changes a protein’s amino acid sequence.

Triplet code —The arrangement of the genetic code such that three DNA bases code for one amino acid.

Aging appears to be largely due to mutagenic oxidants produced as by-products of normal metabolism. These oxidants, such as hydrogen peroxide, are the same mutagens produced by radiation, and cause damage to DNA, proteins, and lipids. The DNA in each cell of a normal rat receives on average about 100,000 oxidative lesions (altered bases) per day. DNA-repair enzymes constantly remove this damage, but they do not keep up: an old rat has over one million oxidative lesions in the DNA of each cell. A human cell receives about ten times fewer lesions than a rat cell, which is consistent with the higher cancer rate and shorter life span of a rat. Decay of mitochondria with time, due to oxidative damage, appears to play a major role in aging. The degenerative diseases of aging, such as cancer, cardiovascular disease, cataracts, and brain dysfunction, are increasingly found to have, in good part, an oxidative origin.

Resources

BOOKS

Bignold, Leon P. Cancer: Cell Structures, Carcinogens and Genomic Instability. New York: Birkhauser, 2005.

Clark, David P. Molecular Biology made Simple and Fun. 3rd ed. St. Louis: Cache River Press, 2005.

Dale, Jeremy W. and Simon F. Park. Molecular Genetics of Bacteria. New York: John Wiley & Sons, 2004.

Louise H. Dickerson

Mutagen

In the living cell , deoxyribonucleic acid (DNA) undergoes frequent chemical change, especially when it is being replicated. Most of these changes are quickly repaired. Those that are not, result in a mutation . Thus, mutation is a failure of DNA repair. Mutagens are chemicals or physical factors (such as radiation ) that increase the rate of mutation in the cells of bacteria , plants, and animals (including humans). Most mutagens are of natural origin and are not just a modern phenomenon.

Mutagens can be found in the food we eat, the air we breath, or the ground we walk on. Very small doses of a mutagen usually have little effect while large doses of a mutagen could be lethal. DNA in the nuclei of all cells encodes proteins , which play important structural and functional (metabolic) roles in the cell. Mutagens typically disrupt the DNA of cells, causing changes in the proteins that the cell produces, which can lead to abnormally fast growth (cancer ), or even cell death . In rare incidences, mutagens can even cause protein changes that are beneficial to the cell.

History

The first mutagens to be identified were carcinogens, or cancer-causing substances. Early physicians detected tumors in patients more than 2,000 years before the discovery of chromosomes and DNA. In 500 b.c., the Greek Hippocrates named crab-shaped tumors cancer (meaning crab).

In England in 1775, Dr. Percivall Pott wrote a paper on the high incidence of scrotal cancer in chimney sweeps who were typically boys small enough to fit inside chimneys and clean out the soot. Pott suggested that chimney soot contained carcinogens that could cause the growth of the warts seen in scrotal cancer. Over a 150 years later, chimney soot was found to contain hydrocarbons capable of mutating DNA.

In France in the 1890s, Bordeaux wine workers showed an unusually high incidence of skin cancer on the back of the neck. These workers spend their days bending over in the fields picking grapes , exposing the back of their necks to the sun . The ultraviolet (UV) radiation in natural sunlight was later identified as a mutagen.

Where mutagens exist

Mutagens can be found in foods, beverages, and drugs. Sometimes a substance is mutagenic because it is converted in the body into something harmful. Regulatory agencies are responsible for testing food and drugs to insure that the public is not unknowingly exposed to mutagens. However, some mutagen-containing substances are not tightly controlled. One such substance is found in the tobacco of cigars and cigarettes.

Some mutagens occur naturally, and some are synthetic. Cosmic rays from space are natural, but they are mutagenic. Some naturally occurring viruses are considered mutagenic since they can insert themselves into host DNA. Hydrogen and atomic bombs are manmade, and they emit harmful radiation. Radiation from nuclear bombs and gaseous particles from nitrogen mustard and acridine orange have been used destructively in war. On the other hand, some mutagens are used constructively to kill bacteria that could grow in human foods, such as the small doses of nitrites used to preserve some meat. Even though nitrites can be mutagenic, without the nitrites these meats could cause botulism ; hence, the benefit outweighs the risk involved.

How mutagens work

Mutagens affect DNA in different ways. Some mutagens, such as nitrogen mustard, bind to a base and cause it to make a different amino acid . These mutagens cause point mutations, because they change the genetic code at one point, so changing a protein's amino acid sequence.

Mutagens such as acridine orange work by deleting or inserting one or more bases into the DNA molecule , shifting the frame of the triplet code for an amino acid. Deletion and insertion mutations causing "frame-shift" mutations can change a long string of amino acids, which can severely alter the structure and function of a protein product.

Normal cells recognize cues from their environment and respond with specific reactions, but cells impaired by a mutation do not behave or appear normal, and are said to be transformed.

Somatic vs. germline mutations

The significance of mutations is profoundly influenced by the distinction between germline and soma. Mutations in somatic (body) cells are not transferred to offspring. Mutations that occur in a somatic cell, in the bone marrow or liver for example, may damage the cell, make the cell cancerous or even kill the cell. Whatever the effect, the ultimate fate of that somatic mutation is to disappear when the cell in which it occurred, or its owner, dies. However, mutated DNA can only be passed to the next generation if it is present in a germ cell such as spermatozoa and ova (eggs), each of which contribute half of the DNA of the new organism . Germline mutations will be found in every cell descended from the zygote to which that mutant gamete contributed. If an adult is successfully produced, every one of its cells will contain the mutation. Included among these will be the next generation of gametes, so if the owner is able to become a parent, that mutation will pass down to yet another generation.

Types of mutagens

Chemical mutagens are classified as alkylating agents, cross-linking agents, and polycyclic aromatic hydrocarbons (PAHs). Alkylating agents act by adding molecular components to DNA bases, which alters the protein product. Cross-linking agents create covalent bonds with DNA bases, while PAHs are metabolized by the human body into other potentially mutagenic molecules.

Radiation is another potent mutagen. For biologists, the most significant forms of radiation are light , heat , and ionizing radiation . Ionizing radiation can penetrate cells and create ions in the cell contents. These, in turn, can cause permanent alterations in DNA; that is, mutations. Ionizing radiation includes: x rays , gamma rays, and the subatomic particles—neutrons, electrons ("beta" particles), and alpha particles (helium nuclei). Ionizing radiation alters the way two strands of DNA interact. This high energy radiation passes through cells and tissues, cutting up any DNA in its path. It can rearrange entire sections of the chromosomes, altering relatively long stretches of DNA. UV radiation causes covalent bonds to form between neighboring thymine bases in the DNA, so altering the DNA product at that location.

Mutagens are often associated with specific cancers in humans. Aromatic amines are mutagens that can cause bladder cancer. Tobacco taken in the form of snuff contains mutagens that can cause nose tumors. Tobacco smoke contains mutagens such as PAHs and nitrosamine (a type of alkylating agent), as well as toxins such as carbon monoxide , cyanide, ammonia , arsenic, and radioactive polonium. Although tobacco products are legal and widely available, many physicians and government agencies warn about the health risks linking smoking with several types of cancer and heart disease .

In 1973, Bruce Ames introduced the most widely-used test to identify potential mutagens. Suspected mutagens are mixed with a defective strain of the bacteria Salmonella, which only grows if it is mutated. Substances that allow the Salmonella to grow are considered mutagenic.

Natural defense against mutagens

In addition to mutagen-induced DNA changes, spontaneous mutations occur in the dividing cells of the human body every day. The nuclei of the cells have repair enzymes, which remove mutations and restore mutated DNA to its original form. If these natural DNA repair mechanisms fail to keep up with the rate of mutation or the repair mechanisms themselves are defective, disease can result. This latter case is seen in lung cancer due to cigarette smoking, where the nicotine in the smoke is thought to block an important repair process in the lungs.

Aging appears to be due, in good part, to mutagenic oxidants produced as by-products of normal metabolism . These oxidants, such as hydrogen peroxide , are the same mutagens produced by radiation, and cause damage to DNA, proteins, and lipids. The DNA in each cell of a normal rat receives on average about l00,000 oxidative lesions (altered bases) per day. DNA-repair enzymes constantly remove this damage, but they do not keep up: an old rat has over one million oxidative lesions in the DNA of each cell. A human cell receives about ten times fewer lesions than a rat cell, which is consistent with the higher cancer rate and shorter life span of a rat. Decay of mitochondria with time , due to oxidative damage, appears to play a major role in aging. The degenerative diseases of aging, such as cancer, cardiovascular disease, cataracts, and brain dysfunction, are increasingly found to have, in good part, an oxidative origin.

It has been argued that dietary antioxidants , such as vitamins C and E and carotenoids, play a major role in minimizing this damage; however, most of the world's population is receiving inadequate amounts of them, at a great cost to health. The main source of dietary antioxidants is fruits and vegetables . Insufficient fruit and vegetable consumption increases the rate of most types of cancer about two fold. A better diet is thought to be a major contributor to the ever increasing life expectancy in the United States and the decreasing (age-adjusted) cancer death rates in non-smokers.

See also Ames test; Carcinogen; Cigarette smoke.

Resources

books

Ruddon, Raymond. Cancer Biology. 2nd ed. New York: Oxford University Press, 1987.

Voet, Donald and Judith Voet. Biochemistry. New York: John Wiley & Sons, 1990.

Woodburn, John. Cancer: The Search for Its Origins. New York: Holt, Rinehart and Winston, Inc., 1964.

Louise H. Dickerson

KEY TERMS

Amino acid

—An organic compound whose molecules contain both an amino group (-NH2) and a carboxyl group (-COOH). One of the building blocks of a protein.

DNA

—Deoxyribonucleic acid; the genetic material in a cell.

Frame shift mutation

—Mutation caused by the deletion or insertion one or more bases into the DNA molecule, shifting the frame of the triplet code for an amino acid. Such mutations can change a long string of amino acids, thereby severely altering the structure and function of a protein product.

Genetic code

—The blueprint for all structures and functions in a cell as encoded in DNA.

Mutation

—A change made to DNA.

Point mutation

—A change in the genetic code at one point, which changes a protein's amino acid sequence.

Triplet code

—The arrangement of the genetic code such that three DNA bases code for one amino acid.

Mutagenesis

Mutagenesis is the process of inducing mutations. Mutations may occur due to exposure to natural mutagens such as ultraviolet (UV) light, to industrial or environmental mutagens such as benzene or asbestos, or by deliberate mutagenesis for purposes of genetic research. For geneticists, the study of mutagenesis is important because mutants reveal the genetic mechanisms underlying heredity and gene expression. Mutations are also important for studying protein function: Often the importance of a protein cannot be characterized unless a mutant can be made in which that protein is absent.

Noninduced Mutagenic Agents

Environmental agents can influence the mutation rate not only by increasing it, but also by decreasing it. For example, antioxidants, which are found commonly in fruits and vegetables, are thought by many to protect against mutagens that are generated by normal cellular respiration. In addition to protective agents, however, many plants also contain deleterious mutagens known as carcinogens . Many chemical mutagens exist both naturally in the environment and as a result of human activity. Benzo(a)pyrene, for example, is produced by any incomplete burning, whether of tobacco in a cigarette or of wood in forest fires.

Spontaneous (noninduced) mutations are very rare, and finding them is difficult because most are recessive . The recessive nature of most mutations means that they will not be evident in most of the individuals who inherit them, for they will be hidden by the presence of the dominant allele. The rarity of mutations means that many individuals must be examined to find a mutant, whether they are people, other organisms, or even cells in culture.

Creating Mutations

To overcome the problem of the rarity of mutations, researchers induce mutations with a variety of agents. Hermann Muller was the first to do this when, in 1927, he used X rays on fruit flies (Drosophila ) to increase the mutation rate by more than 100-fold. Other high-energy forms of radiation can also be used to create mutations.

The first chemical to be recognized as a mutagen was mustard gas, which had been developed during World War I, but not tested until World War II by Auerbach and Robson, at the University of Edinburgh. Since then a wide variety of chemicals have been discovered that are also mutagenic. Some induce mutations at any point in the cell cycle, by disrupting DNA structure. Others only act during DNA replication. Called base analogs, these latter chemicals have structures similar to the bases found in DNA, and are incorporated instead of the normal base.

Transposable genetic elements (also called transposons, or "jumping genes") can also induce mutations. These elements insert randomly into the genome, and may disrupt gene function if inserted into a gene or its promoter. Finding the organism with a disrupted gene is made easier if the transposon carries with it a reporter gene whose product can be identified, or a selectable marker that allows the transformed cells to live while non-transformed ones die. (The use of reporter genes and selectable markers are techniques used in genetic analysis in the laboratory.) The transposon sequence itself serves as a molecular tag. Thus, if the target gene (the gene being studied) is interrupted, finding the transposon allows the researcher to find the gene.

All of the above methods disrupt genes randomly. However, specific genes can also be targeted, for "site-directed mutagenesis," if their sequence is known. Using the known sequence, a matching DNA sequence is inserted into a single-stranded vector . Short, complementary, partial sequences containing the desired mutation are then synthesized. These are allowed to pair up, and DNA polymerase is then used to complete the complementary strand. Further replication amplifies he number of copies of the mutant. In bacteria, the mutant gene can be placed on a plasmid for transformation of the bacteria. The bacteria make the mutant protein, and the effect of the mutation can then be studied. This is a key tool in studying how amino acid sequences affect protein structure, since individual amino acids can be changed, one at a time.

In eukaryotes, the mutant gene can be inserted into the chromosome of an experimental organism by "homologous recombination," a system in which the mutant gene switches places with the normal chromosomal gene. Such techniques can "knock out" and "knock in" genes bearing the desired mutations.

The First Mutagenesis Assay

Before DNA sequencing became widespread, most mutations could only be detected by their effects on the phenotype of the organism. Many mutations are recessive, however, and do not affect the phenotype if present in only one allele. Hermann Muller, who pioneered the study of mutations, overcame this problem by focusing on the X chromosome in his studies of the genetics of Drosophila, the fruit fly. While females have two X chromosomes, males have only one, so any mutated gene carried on the X chromosome is expressed in males, even if it is recessive. Hence recessive lethal mutations on the X chromosome kill any male inheriting them, but would not kill a female. Muller's method examined all of the genes on the X chromosome that could mutate to give a recessive lethal mutation. Muller used X rays to generate mutants. X rays are a very high-energy form of radiation, and break the DNA at numerous points. The method is shown in Figure 1.

Muller treated adult males with X rays and mated them to females who carried one copy of a specially prepared X chromosome, called ClB. This chromosome had a gene to prevent crossing over (C ), which kept the chromosome intact; a lethal recessive gene (l ) to kill any males that inherit it; and a dominant "bar eye" gene (B ) that resulted in a distinctive phenotypic change, making it easy to find female flies that inherited it.

Muller mated X-ray treated males with ClB -carrying females. All female offspring from this crossbreeding received one treated X chromosome from the male (which might or might not have carried a lethal recessive gene). They also received one X from the female, either normal or ClB. He selected only the bar-eyed females for further mating. To determine which of these females carried an X-ray induced lethal recessive, he separated each female into a separate jar, and examined their offspring.

Three types of males were created in this cross, depending on what type of X chromosome they inherited. Males inheriting the ClB chromosome died, due to the presence of the l gene. Males inheriting an X-ray-treated X chromosome with a lethal recessive died. Males inheriting an X-ray-treated X chromosome without a lethal recessive lived. Therefore, any jar with live males indicated that the mother did not carry a lethal recessive. Any jars with no males indicates the mother carried a lethal recessive, originally induced in the X-ray-treated male. The analysis was rapid because an experienced person could examine a bottle of flies and see at a glance if there were males present. Subsequent studies showed that this method tested almost 1,000 genes simultaneously, thus making it practical to use when detecting rare mutations. Unfortunately the breeding takes quite a lot of time, so this assay has now fallen largely into disuse, despite its historical importance.

Detecting Mutations

Today the mutagenic potential of chemicals is considered in evaluating the mutagenic risks posed by chemical exposure. Many new methods have been developed to determine if chemicals to which people will be exposed, such as new drugs, food additives, and pesticides, are mutagens. Since mutations can occur in any organism, and because there are many different kinds of mutations, there are a correspondingly wide variety of tests to detect them. No one test detects them all.

The Ames test was the first and remains the only test to be almost universally required by regulatory agencies as a minimum standard for determining if a chemical is mutagenic. The test is conducted in Salmonella bacteria. Since bacteria have only one chromosome, recessive mutations can be detected readily. Rare mutations are easily detected because mutants can be selected very simply. Several variants have been added to the original test, allowing for detection of many types of mutations. In an effort to make the test more relevant to human risk, one variant uses an extract of liver to mimic the biochemical modifications of chemicals that occur in the human liver.

Assays for Chromosome Aberrations.

Chromosomal aberrations can be detected by examining cells in mitosis or meiosis for changes (see Figure 2). Typically, bone marrow cells of mice or rats are examined for in vivo tests. Any cells can be used for tests of cells in culture, but Chinese hamster cells or human fibroblasts are most commonly used. Another test, called the micronucleus test, is also commonly used. Micronuclei are small nuclei that arise from pieces of chromosomes or whole chromosomes that have been lost during cell division. They are conveniently detected in mouse red blood cells, which have no normal nucleus but which often retain micronuclei. The micronucleus assay is also widely used in cultured cells.

Assays for Somatic Mutations.

Recessive mutations can be detected more readily on a mammalian X chromosome than on the other chromosomes, because only one X chromosome is active. Therefore, detection of the mutagenic potential of a substance in mammals can be most efficiently performed by analyzing the X-linked mutations. A system using the X-linked gene hprt has been widely used because the enzyme is not essential and because the addition of the drug thioguanine kills all cells except mutants. A count of the cells that can be cultured in the presence of thioguanine is a count of hprt mutants.

see also Ames Test; Mutagen; Mutation; Mutation Rates.

John Heddle

Bibliography

Griffiths, Anthony J. F., et al. An Introduction to Genetic Analysis. New York: W. H.Freeman, 2000.

Muller, Hermann J. "Artificial Transmutation of the Gene." Science 66 (1927): 84-87.

Rubin, G. M., and A. C. Spradling. "Genetic Transformation of Drosophila withTransposable Element Vectors." Science 218 (1982): 348-353.

Internet Resource

United Nations Scientific Committee on the Effects of Atomic Radiation. <http://www.unscear.org/>.

Michael Smith of Canada received the 1993 Nobel Prize in physiology or medicine for invention of site-directed mutagenesis. He shared the prize with Kary Mullis, who invented the polymerase chain reaction.

Mutagenesis

Resources

Mutagenesis is an induced genetic change in a cell by the alterations in the cell’s genetic material (usually deoxyribonucleic acid (DNA)). This alteration can be inherited and passed to subsequent generations. While many mutations are benign, some can be detrimental and cause human genetic disease. If the mutation occurs in a gamete (sex cell), the genetic alteration may be passed to subsequent generations. Mutations provide a mechanism for evolution if the mutations in the DNA produce a new or modified protein that has enhanced or new beneficial functions such that this newly acquired characteristic has a selective survival advantage and thus will be more likely to be passed down from one generation to the next. Mutations in somatic cells, or cells that have undergone differentiation and will not have the potential to develop into any other cell types are usually spontaneous and not inherited.

Mutagenesis can result in alteration of an individual gene, or a specific letter in the DNA alphabet called a nucleotide. There are four nucleotides; adenine, gua-nine, cytosine, and thymine. Mutagenesis—the process acquiring mutations—can be in the form of a point mutation (a mutation at a single nucleotide), a deletion of one or more nucleotides, or a duplication in the DNA sequence. A point mutation can cause a genetic disease if it occurs in a gene important for normal cellular function. Deletions involve deleted sequence of DNA that can be in a coding gene sequence (called an exon) or a noncoding sequence of the gene (called an intron) or the noncoding sequence that separates genes. Deletions can be in a single nucleotide or span several genes. A duplication involves a sequence of DNA that gets repeated.

Mutagenesis can occur as the result of exposure to ionizing radiation and certain chemicals. Ionizing radiations include cosmic rays, x rays, and ultraviolet light. Melanoma, which is caused almost exclusively by exposure to the ultraviolet radiation from the sun, is the most rapidly increasing lethal cancer in the United States. In melanoma, ultra violet radiation induces what is called thymine dimers, where two thymine nucleotides next to each other in a DNA

KEY TERMS

Deoxyribonucleic acid (DNA) —The genetic material in a cell.

Genotype —The full set of paired genetic elements carried by each individual, representing the its genetic blueprint.

Mutation —Molecular defect in DNA.

Nucleotide —Molecular unit that is the building block of DNA.

Phenotype —The outward manifestation of the genotype, including an organism’s morphological, physiological, and many behavioral attributes.

sequence form an abnormal bond. Thymine dimers in a skin cell can lead to skin cancer. The heightened prevalence of melanoma is presumably due to increased ultraviolet radiation exposure of skin most likely from depletions in the ozone layer.

Mutations that affect tumor suppressor genes can result in uncontrollable cellular growth, also called cancer. If this cancer is not detected early and removed, it can become vascularized and leak into the bloodstream. If it metastizes in this way, the cancer can quickly spread to other organs. Additionally, a number of chemicals have been identified as mutagenic such as the common household cleaner bleach. Many chemicals intercalate into the DNA double helix and cause errors during DNA replication. Chemicals that bind to DNA are called DNA adducts. There are many environmental carcinogens or cancer-causing chemicals that induce mutagenesis.

Teratogenesis is the development of congenital abnormalities due to the exposure to substances during development called teratogens. Many teratogens are mutagens in that they can cause mutations in DNA. An example of a teratogen is thalidomide. Thalidomide was used in the 1950s to as a treatment for morning sickness in pregnant women. It was later found to cause birth defects. In this case, it disrupted developmental processes resulting in abnormalities affecting normal development.

Despite the many different ways that mutagenesis can occur, mechanisms for DNA repair exist that help maintain the integrity of the genome. One mechanism, called nucleotide excision repair, involves recognition and removal of damaged DNA by nicking it where there is a point mutation, removing the damaged nucleotide sequence, re-synthesis to add the correct nucleotide, and DNA ligation of the nicked DNA to seal the DNA where it is being repaired.

Resources

BOOKS

Bignold, Leon P. Cancer: Cell Structures, Carcinogens and Genomic Instability. New York: Birkhauser, 2005.

Clark, David P. Molecular Biology made Simple and Fun. 3rd ed. St. Louis: Cache River Press, 2005.

Dale, Jeremy W. and Simon F. Park. Molecular Genetics of Bacteria. New York: John Wiley & Sons, 2004.

Bryan Cobb

Mutagen

A mutagen is any substance or agent that can cause a mutation, or change in the sequence or structure of DNA. Mutagens are classified on the basis of their physical nature and the types of damage they do. A mutagen is not the same as a carcinogen. Carcinogens are agents that cause cancer. While many mutagens are carcinogens as well, many others are not. The Ames test is a widely used test to screen chemicals used in foods or medications for mutagenic potential.

Chemical Mutagens

There are many hundreds of known chemical mutagens. Some resemble the bases found in normal DNA; others alter the structures of existing bases; others insert themselves in the helix between bases; while others work indirectly, creating reactive compounds that directly damage the DNA structure.

"Base analogs" are molecules whose chemical structure is similar to one of the four DNA bases (adenine, thymine, cytosine, and guanine). Because of this similarity, they can be incorporated into the helix during DNA replication. A key feature of mutagenic base analogs is that they form base pairs with more than one other base. This can cause mutations during the next round of replication, when the replication machinery tries to pair a new base with the incorporated mutagen. For instance, 5-bromo-deoxyuridine (5BU) exists in two different forms. One mimics thymine and therefore pairs with adenine during replication, while the other mimics cytosine and therefore pairs with guanine. In its thymine-mimicking form, 5BU can be incorporated across from an adenine. If it then converts to its cytosine-like form, during the next round of replication, it will cause a gua-nine to enter the opposite strand, rather than the correct adenine.

"Base-altering mutagens" cause chemical changes in bases that are part of the DNA. For example, nitrite preservatives in food convert to the mutagen nitrous acid. Nitrous acid causes deamination, or loss of an-NH₂ group, of cytosine. When this occurs, cytosine becomes uracil, a base that is not normally incorporated in DNA but that is very similar to thymine. Unless repaired, this uracil will cause an adenine to enter the opposite strand instead of a guanine. Many base-altering mutagens are complex organic molecules. These are formed in large quantities in smoke, making up the "tar" of cigarette smoke, for example. They act as alkylating agents, combining with DNA to form bulky groups that interrupt replication.

"Intercalating agents" are flat molecules that insert themselves between adjacent bases in the double helix, distorting the shape at the point of insertion. Where this occurs, DNA polymerase may add an additional base opposite the intercalating agent. If this occurs in a gene, it induces a frameshift mutation (that is, it alters the reading of the gene transcript, changing which amino acids are added to the encoded protein). Ethidium bromide is one such agent, widely used in DNA research because its dark color allows DNA to be easily visualized. This is useful in gel electrophoresis, for instance, to find the DNA bands that have been separated in a gel.

Other damaging agents include chemicals that create "free radicals" inside a cell. Free radicals are compounds in which an atom, usually an oxygen, has an unbonded electron. Free radicals are highly reactive and can cause several types of damage to DNA.

Light and Radiation

Radiation refers to two different phenomena: light and high-energy particles. Visible light represents a small slice of the electromagnetic spectrum, which includes long-wavelength (low-energy) radio waves and short-wave-length (high-energy) ultraviolet waves, plus X rays and gamma rays. These high-energy forms can directly disrupt DNA by breaking its chemical bonds. In severe cases, this can break apart chromosomes, causing chromosome aberrations. More often, they create mutagenic free radicals in the cell. X rays were first used by Hermann Muller to induce mutations in fruit flies. They continue to be used to create mutations in model organisms to study genes and their effects.

Ultraviolet light is less energetic than X rays but causes mutations nonetheless. The higher-energy form, UV-B, is more toxic than UV-A, because of its potential to cause cross-linking between adjacent thymine or cytosine bases, creating a so-called pyrimidine dimer (cytosine and thymine are chemically classified as pyrimidines). Pyrimidine dimers interrupt replication. UV-A does not cause dimer formation but can still cause mutations by creating free radicals.

Another meaning of the term "radiation" is high-energy particles released during the breakdown of radioactive elements, such as uranium. These particles are either electrons (called beta particles) or helium nuclei (called alpha particles). Their energy is sufficient to disrupt DNA structure, or to create free radicals.

Repairing the Damage

DNA is constantly being damaged, and it is constantly being repaired as well. It is only when the damage is not repaired that a mutation can lead to cancer or cell death. The DNA repair enzymes can recognize damaged nucleotides and remove and replace them. The human liver contains a large number of enzymes whose role is to detoxify toxic compounds, mutagenic or otherwise, by chemically reacting them. However, in some cases these enzymes (called cytochrome P450s) actually create mutagens during the course of these reactions. Such "bioactivation" may be a significant source of mutagens.

see also Carcinogens; Chromosomal Aberrations; DNA Repair; Muller, Hermann; Mutagenesis; Mutation.

Richard Robinson

Bibliography

Philp, Richard B. Ecosystems and Human Health: Toxicology and Environmental Hazards, 2nd ed. Boca Raton, FL: Lewis Publishers, 2001.

Brusick, David. Principles of Genetic Toxicology, 2nd ed. New York: Plenum Press, 1987.