Chapter 15 Mutagenic Pollutants 15.1 INTRODUCTION A mutation is a process by which the hereditary constitution of a cell is altered, ultimately resulting in a genetically altered population of cells or organism. Although mutations can occur in the RNA of viruses and the DNA of cytoplasmic organelles, the mutations of greatest interest occur within genes in the nucleus of the cell. The human body is estimated to contain more than 10 trillion cells, and at some stage in its life cycle each cell contains a full complement of the genes needed by the entire organism. Genes, composed of DNA in the nucleus of cells, are clustered together in chromosomes. In the chromosomes of all but the most primitive organisms, DNA is combined with protein. DNA, the molecular basis of heredity in higher organisms, is made up of a double helix held together by hydrogen bonds between purine and pyrimidine bases, i.e., between adenine (A) and thymine (T), and between guanine (G) and cytosine (C). Figure 15.1 shows the structures of the five bases in DNA and RNA, and the pairing of bases in DNA is shown in Figure 15.2. The highly specific complementarity of these bases enables DNA to act as a template for its replication by DNA polymerases, a s well as the synthesis of RNA transcripts by RNA polymerases. For the information contained in DNA to be biologically expressed, the sequence of the nucleotides in a gene is converted into the sequence of amino acids in a protein. It is the amino acid sequence that determines the enzymatic and structural properties of the protein thus formed. DNA clearly plays a pivotal role in the expression and perpetuation of life. However, it is also a critical target for the action of many mutagenic environmental chemicals; lesions in DNA may occur through the action of physical or chemical agents found in the environment. Occurrence of mutation, however, depends on the nature of the initial lesion and the response of cells to the DNA damage. If the damage is intermediate, the mutations resulting from [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 269 269-278 FIGURE 15.1 Structures of bases in nucleic acids. # 2005byCRCPressLLC it may be of immediate concern because mutations are implicated in pathogenesis of many inherited, somatic human diseases. However, if the damage is severe enough it can interfere with essential functioning of DNA and lead to the death of cells. 15.2 TYPES OF MUTATION Mutations are often divided into two broad categories. The first category is chromosomal aberration, which refers to mutations that are cytologically visible. The second is gene mutation, mutations that occur at the submicro- scopic level and are cytologically invisible. 15.2.1 C HROMOSOMAL ABERRATIONS A human cell normally has 23 pairs of autosomal chromosomes and a pair of sex chromosomes. In chromosomal aberration, mutation produces either a change in the number of chromosomes or a change in the structure of individual chromosomes. Changes that involve entire sets of chromosomes are called euploidy, whereas variations that involve only single chromosomes within a set are called aneuploidy. Alteration in chromosomal structure occurs when the chromosomes fracture and the broken ends rejoin in new combinations. Major structural changes include deletions, duplications, inversions, and translocations. In deletion, a portion of a chromosome is lost (e.g., in ABCDE, the portion C is lost, becoming ABDE), whereas in duplication, an additional copy of a portion of the chromosome is inser ted (e.g., ABCCDE). Deletions and duplications both upset the metabolic balance of an organism by altering the amount of gene product formed. An inversion is when the order of genes on a chromosome is reversed in one area (e.g., ABCDE becomes ACBDE). If a broken portion of a chromosome attaches itself to a second chromosome, it is termed a translocation (e.g., ABCDE ! ABDE þ C, C þ ABC ! ABCC). Because the position of a gene affects its regulation and activity, inversions and translocations may be detrimental. 270 Environmental Toxicology [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 270 269-278 FIGURE 15.2 Pairing of bases in DNA. # 2005byCRCPressLLC 15.2.2 GENE MUTATIONS In a gene mutation, an alteration occurs in the nucleotide sequence of a gene, which cannot be observed microscopically. Two subclasses of gene mutations have been identified: point mutations and intragenic deletions. Point mutations may involve the displacement of one nucleic acid base by another (base-pair substitution), resulting in substitution of one amino acid for another in the final gene product, thus altering cellular function. Alternatively, they may involve insertion or deletion of a nucleotide or nucleotides within a polynucleotide sequence of a gene (frameshift mutations). This leads to alteration in the nucleotide sequence, thus producing an incorrect gene product. An intragenic deletion occurs when a more-extensive deletion occurs within a gene, so that the infor mational material of that gene is essentially lost. 15.3 EFFECT OF MUTATIONS Mutations often induce deleterious effects on the individuals or populations affected. While the effects of several individual mutagens (agents that cause mutations) are discussed later in this chapter, a general concept is addressed here. One of the concerns over mutagenic environmental agents is their relationship with cancer. As is widely recognized, the majority of human cancers appear to be related to environmental factors, and man y mutagens have been shown to be carcinogens (cancer-causing agents). However, in the long term, the ability of different environmental agents to cause mutations (and teratogenic effects) may create a greater burden on society than cancer does because of the increased incidence of genetic disease and birth defects. The total impact of genetic disease on national health is unknown. Autosomal dominant disorders have been shown to occur in 8 of 10,000 births. 1 A newspaper in British Columbia, Canada, reported that 9.4 individuals out of every 100 live births suffer from genetic diseases or disabilities, and that 2.7 of every 100 live births have disorders of unknown etiology that may be partly genetic. Serious consequences can result if a mutation occurs in such a way that a hydrophilic amino acid is substituted for a hydrophobic residue in the resultant protein, or vice versa. Sickle-cel l anemia, a hereditary disease, is a typical example. This disease is the result of a biochemical lesion caused by substitution of glutamic acid (a hydrophilic amino acid) for valine (a hydrophobic amino acid) in a chain of approximately 140 amino acids in human hemoglobin. This seemingly minor change produces abnormally shaped red blood cells that can no longer transport oxygen efficiently, leading to detrimental anemia. Conversely, mutations may not necessarily produce deleterious effects on an organism. For instance, if a mutation occurs in such a way that only one amino acid along the backbone of a protein is incorrectly specified, the three- dimensional structure of the protein may not be greatly altered, allowing it to Mutagenic Pollutants 271 [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 271 269-278 # 2005byCRCPressLLC function properly. This is usually the case when a hydrophilic amino acid residue in a protein is replaced by another hydrophilic amino acid, or a hydrophobic–hydrophobic replacement occurs. Occasionally, a mutation may occur that results in the ability of a cell or a species to survive being improved. However, humans are highly developed organisms, and so when a mutation does occur, the probability is that it will be a deleterious one. 15.4 INDUCTION OF MUTATION Commonly found mutagens that are of most concern to humans include: ultraviolet (UV) light, ionizing radiation, microtoxins, and organic and inorganic chemicals. Some common environmental mutag ens and their sources are listed in Table 15.1. 15.4.1 UV L IGHT The region of the electromagnetic spectrum with wavelengths between 200 and 300 nm is of primary biological importance. The main reason for this is that DNA absorbs most strongly at 260 nm. It has been shown that mutations in microorganisms can be caused by irradiation of growth medium by UV light. Production of mutations by UV light, however, is strongly influenced by repair processes that reverse or remove induced photoproducts in DNA. One of the most important ways in which the biological activity of DNA is altered by UV irradiation is thymine dimerization, a reaction in which two thymine molecules are fused together to form a dimer (Figure 15.3a). This dimerization may occur between adjacent thymine residues, or between two thymine residues across the chains (interchain dimerization). Dimerization results in disruption of hydrogen bonding between the bases in the DNA molecule (Figure 15.3b). Chain break (P - S - P - S) is another possible result. UV irradiation can also cause hydration of cytosine (Figure 15.4), which may also result in hydrogen-bond disruption. The effect of UV irradiation is not 272 Environmental Toxicology [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 272 269-278 Table 15.1 Common Environmental Mutagens Mutagen Sources UV light Sunlight Ionizing radiation Cosmic rays, medical x-rays Nitrosamines Pyrolysis products of tryptophan, broiled meat, beer and whisky Benzo[a]pyrene Cigarettes and wood smoke Benzidine Textile dyes, manufacture of paper and leather Cr(VI), Hg Metal alloys, mines Hydrazine Cigarettes and wood smoke Malonaldehyde Peroxidized polyunsaturated fatty acids Vinyl chloride Plastics Aflatoxin B 1 Fungi-contaminated grains and peanut # 2005byCRCPressLLC limited to DNA. Proteins and RNA outside the nucleus and other cellular components may also be affected. 15.4.2 I ONIZING RADIATIONS Examples of ionizing radiations include x-rays, g-rays, a-particles, high-energy neutrons, and electrons. Ionizing radiation produces various kinds of DNA damage, such as altering DNA bases, or producing single- or double-strand breaks in the phosphodiester chains of the DNA molecule, leading to frangmentation of the DNA. Such damages will consequently change the coding properties of DNA, resulting in induction of mutations. Mutagenic Pollutants 273 [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 273 269-278 FIGURE 15.3 (a) UV radiation-initiated formation of a thymine dimer, and (b) interchain dimerization disrupts hydrogen bonding between DNA bases. FIGURE 15.4 Hydration of cytosine. (a) (b) # 2005byCRCPressLLC 15.4.3 CHEMICAL MUTAGENS Approximately 70,000 commercial chemicals are in use in the U.S., and this number is increasing by 1000 new compounds each year. 2 There are also many environmental chemicals that are of concern. Some of these are derived from the commercial chemicals, while others are produced from anthropogenic sources. Anthropogenic sources include: industrial processes involving com- bustion of fossil fuels, transportation, 3 open burning of scrap rubber tires, combustion of agricultural wastes (such as sugar cane, orchard prunings, and grain straws), municipal sewage sludges, 4 herbicide such as S-(2-chloroallyl) diethyldithiocarbamate (sulfallate), 5 and textile manufacturing. Mutagenic compounds have been classified into seven major categories, based on their actions on DNA. The categories are: 6 alkylation arylation intercalation base analog incorporation metaphase poisons deamination enzyme inhibition Table 15.2 summarizes the mechanisms involved in these categor ies. Some examples are given in the following sections. 15.4.3.1 Alkylating Agent s Alkylating agents represent the largest group of mutagens. They may carry 1, 2, or more alkyl groups in a reactive form, and thus are called mono-, bi-, or polyfunctional alkylating agents. These compounds can cause base alkylation, depurination, backbone breakage, or alkylation of phosphate groups. For example, most nitr oso compounds are highly mutagenic (and carcinogenic) because of their ability to form electrophilic species. Figure 15.5 gives an 274 Environmental Toxicology [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 274 269-278 Table 15.2 Mechanisms of Action of Several Mutagenic Agents Chemical action Mechanism of action Alkylation Addition of an alkyl group (CH 3 CH 2 CH 2 –, etc.) to a nucleotide Arylation Covalent bonding of an aryl group Intercalation The compound ‘‘wedges’’ into the DNA helix Base analog incorporation Base-pairing errors due to incorporation mispairing Metaphase poisons Interference with spindle formation and disruption of migration and segregation of chromosomes Deamination Removal of an amino group (NH 2 ) from adenine, cytosine, or guanine Enzyme inhibition Interference with biosynthesis of purines or pyrimidines and interference with repair # 2005byCRCPressLLC example showing how diethylnitrosamine, a nitroso compound, can act as an alkylating agent. In this case, diethylnitrosamine is converted into two species, one of which is carbonium CH 3 CH 2 þ ion. This ion may seek such nucleophilic sites as –N– or –S– on informational macromolecules, resulting in the covalent alkylation of a DNA base. For example, N-2 and N-3 of guanine (G) are highly susceptible to electrophilic attack. An alkylated G may not base-pair properly, or the information content of the molecule is altered in some way by the mutation. For instance, the alkylated G pairs with T instead of with C, thus causing transitional-type mutations. It is also possible for the alkyl group of N-7 to labilize the b-glycoside bond, resulting in depurination and leading to transition or transversion. Some chemical mutagens, such as HNO 2 , can react directly with nitrogenous bases of DNA. Other mutagens have structures that are similar to the structure of one of the bases; these are called base analogs. It is possible for these base analogs to be incorporated into a DNA molecule. For example, 5-bromouracil, in its normal (keto) form, hydrogen bonds wi th A (as would U or T), but in its enol form it base-pairs with G. 15.4.3.2 Intercalating Agents Many planar aromatic hydrocarbons are thought to be able to position themselves (intercalate) between the flat layers of hydrogen-bonded base pairs in the interior of the DNA double helix , forcing it to partially uncoil. Such compounds are often called intercalating agents, and as a result of their action errors occur in the transmission of the genetic co de. The chemical structures of several intercalating agents are given in Figure 15.6, and Figure 15.7 illustrates damage to DNA that can be induced by several of the agents discussed so far. 15.4.3.3 Metals Many studies have shown the cytotoxic effects of a variety of metallic salts, which result in the denaturation of macromolecules. The reactions of metallic ions with nucleic acids are particularly important as some of the metals can contribute to mutagenesis and carcinogenesis. As noted in Chapter 12, exposure to mercury (Hg) results in decreased DNA content in cells. Hg also adversely affects chromosomes and mitosis, leading to mutagenesis. The crucial factors in the toxic action of metals such as Hg may involve specific reactions with certain chemical groups in biomolecules, or with certain Mutagenic Pollutants 275 [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 275 269-278 FIGURE 15.5 Diethylnitrosoamine, an alkylating agent. # 2005byCRCPressLLC sites in tissues or organelles. Examples are given in Chapter 12, showing the interaction of Hg and Pb with the –SH group in proteins. A specific example showing the interaction of Pb with d-aminolevulinic acid dehydratase (ALAD) in heme synthesis is also presented. As already noted, some toxic metals can compete with essential metals, such as magnesium (Mg), calcium (Ca), or zinc (Zn). These essential metals are required as cofactors in a number of enzyme systems; or they may contribute to stabilizing the structure of biomolecules . 276 Environmental Toxicology [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 276 269-278 FIGURE 15.6 Examples of intercalating agents. FIGURE 15.7 Mechanisms of DNA damage induced by various agents. # 2005byCRCPressLLC Research has shown that different metallic ions react with different ligands. 7 Mg 2þ and Ca 2þ ions, for example, bind to phosphate groups on nucleotides and tend to stabilize the DNA double helix, whereas Hg and silver (Ag) bind to bases, lowering the stability of the helix. Several studies have shown that chromium (Cr)(VI) compounds induce chromosome aberrations and mutations in cultured mammalian cells. 8,9 Induction of DNA single-strand breaks and DNA–protein crosslinks by Cr(VI) compounds has also been report ed. 10 Cr(VI) compounds can also inhibit the activity of such enzymes as glutathione reductase in cultured cells. After it enters the cell, Cr(VI) is reduced to Cr(III), through the intermediates Cr(V) and Cr(IV). This reduction process is accompanied by the formation of radical species such as active oxygen 11 as well as glutathionyl radicals. 12 These are considered to be responsible for the observed chromate-induced DNA damage. Interestingly, pretreatment with a-tocopherol (vitamin E) was found to reduce Cr-induced chromosomal aberrations. It is thought that because vitamin E is an efficient free-radical scavenger it may scavenge Cr(V) and free radicals. 10 15.5 REFERENCES 1. Stryer, L., Biochemistry, 3rd Ed., W. H. Freeman & Co. Publishers, San Francisco, 1988, p.675. 2. Ames, B., Identifying environmental chemicals causing mutations and cancer, Science, 204, 387, 1979. 3. Pierson, W. R. et al., Mutagenicity and chemical characteristics of carbonac- eous particulate matter from vehicles on the road, Environ. Sci. Technol., 17, 31, 1983. 4. Babish, J.G., Johnson, B.E. and Lisk, D.J., Mutagenicity of municipal sewage sludges of American cities, Environ. Sci. Technol., 17, 272, 1983. 5. Rosen, J.D. et al., Mechanism for the mutagenic activation of the herbicide sulfallate, J. Agric. Food Chem., 28, 880, 1980. 6. Graedel, T. E., Hawkins, D. T. and Claxton, L. D., Atmospheric Chemical Compounds: Sources, Occurrence, and Bioassay, Academic Press, New York, 1986, 35. 7. Jacobson, K.B. and Turner, J.E., The interaction of cadmium and certain other metal ions with proteins and nucleic acids, Toxicol., 16, 1, 1980. 8. Majone, F. and Levis, A.G., Chromosomal aberrations and sister chromatic exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds, Mutation Res., 67, 231, 1979. 9. Tsuda, H. and Kato, K., Chromosomal aberrations and morphological transformation in hamster embryonic cells treated with potassium dichromate in vitro, Mutation Res., 46, 87, 1977. 10. Sugiyama, M., Lin, X. and Costa, M., Protective effect of vitamin E against chromosomal aberrations and mutation induced by sodium chromate in Chinese hamster V79 cells, Mutation Res., 260, 19, 1991. Mutagenic Pollutants 277 [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 277 269-278 # 2005byCRCPressLLC 11. Kawanishi, S., Inoue, S. and Sano, S., Mechanism of DNA cleavage induced by sodium chromate (VI) in the presence of hydrogen peroxide, J. Biol. Chem., 26, 5952, 1986. 12. Shi, X. and Dalal, N.S., Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalized reduction of chromium (VI), Biochem. Biophys. Res. Commun., 163, 627, 1989. 15.6 REVIEW QUESTIONS 1. Define the term ‘‘mutation.’’ 2. How are chromosomal aberrations different from gene mutations? 3. Match the following: A. (1) Inversion (2) Deletion (3) Translocation (4) Duplication B. (a) A portion of a chromosome is lost (b) The order of genes on a chromosome is reversed in one area (c) An additional copy of a portion of the chromosome is inserted (d) A broken portion of a chromosome attaches itself to a second chromosome 4. Which is more deleterious to an animal or a person? (a) Substitution of a hydrophobic amino acid with another hydrophobic acid (b) Substitution of a hydrophilic amino acid for a hydrophobic amino acid. 5. How does UV radiation affect DNA? 6. How do ionizing radiations affect DNA bases? 7. Briefly explain the phenomenon of dimerization. Which environmental agent(s) can cause it? 8. Describe alkylation as a mechanism of mutation induction. 9. Give an example to explain the term ‘‘intercalation.’’ 10. How does Hg interact with the DNA helix? 11. Which is more toxic, Cr(III) or Cr(VI)? Why is Cr mutagenic? 12. Vitamin E appears to reduce the toxicity caused by Cr(VI). What is the possible mechanism involved in this phenomenon? 278 Environmental Toxicology [16:51 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/4365-015.3d] Ref: 4365 MING-HO YU Chap-015 Page: 278 269-278 # 2005byCRCPressLLC . hydrogen-bond disruption. The effect of UV irradiation is not 272 Environmental Toxicology [1 6:5 1 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/436 5-0 15. 3d] Ref: 4365 MING-HO YU Chap- 015 Page: 272 26 9-2 78 Table. induction of mutations. Mutagenic Pollutants 273 [1 6:5 1 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/436 5-0 15. 3d] Ref: 4365 MING-HO YU Chap- 015 Page: 273 26 9-2 78 FIGURE 15. 3 (a) UV radiation-initiated. an 274 Environmental Toxicology [1 6:5 1 26/8/04 P:/CRC PRESS/4365 MING-HO.751 (1670)/436 5-0 15. 3d] Ref: 4365 MING-HO YU Chap- 015 Page: 274 26 9-2 78 Table 15. 2 Mechanisms of Action of Several Mutagenic