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209 CHAPTER 14 Mutagenic Pollutants 14.1 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 organisms. 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 contains a full complement of the genes needed by the entire organism. Genes, composed of DNA in the nucleus, are clustered together in chromosomes. In the chromosomes of all but the most primitive organisms DNA is combined with protein. DNA is the molecular basis of heredity in higher organisms and 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 14.1 shows the chemical structures of the five bases in DNA and RNA. The pairing of bases in DNA is presented in Figure 14.2. The highly specific complementarity of these bases enables DNA to act as a template to direct its replication by DNA polymerases, as 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 of a gene is converted to 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. Clearly, DNA 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, i.e., lesions in DNA may occur through the action of physical or chemical agents found in the environment. Occurrence of mutations, however, depends on the nature of the initial lesion and the responses of cells to DNA damage. If the damage LA4154/frame/C14 Page 209 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC 210 ENVIRONMENTAL TOXICOLOGY is intermediate, the mutations resulting from it may be of immediate concern because mutations are implicated in the pathogenesis of many inherited, somatic human disease states. On the other hand, if the damage is gross enough it can interfere with the essential functioning of DNA and may lead to the death of cells. 14.2 TYPES OF MUTATION Mutations are often divided into two broad categories. One of them is chromo- somal aberration, which refers to mutations that are cytologically visible. The other is called gene mutation, which is cytologically “invisible” mutation that occurs at the submicroscopic level. 14.2.1 Chromosomal 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 chromo- somes. Changes that involve entire sets of chromosomes are called euploidy , whereas variations in number 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 ABC DE, the portion C is lost), whereas in duplication, an additional copy of a portion of the chromosome is inserted (e.g., ABCCDE). Deletions and duplications both upset the metabolic balance of an Figure 14.1 Structures of bases in nucleic acids. Figure 14.2 Pairing of bases in DNA. 1 1 1+ + 2 1 1 + 2 2 1 1 + 2 2 &+ $GHQLQH$*XDQLQH*&\WRVLQH&8UDFLO87K\PLQH7 1 1 1 1 1+ + 2 1 1 1 1 1 + + + + + &+ 1 +1 1 2 6XJDU 2+ 1 + 1 1 1 6XJDU 7K\PLQH $GHQLQH 1 1 2 6XJDU 1 +2 + +1 1 1 1 1 6XJDU &\WRVLQH *XDQLQH + LA4154/frame/C14 Page 210 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC MUTAGENIC POLLUTANTS 211 organism by altering the amount of gene products formed. When the order of genes on a chromosome is reversed in one area, it is called an inversion (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 + C; C + ABC → ABCC). Since the position of a gene affects its regulation and activity, inversions and translocations may be detrimental. 14.2.2 Gene Mutations In a gene mutation, the alteration occurs in the nucleotide sequence of a gene and cannot be observed microscopically. Two subclasses of gene mutations have been identified, i.e., point mutations and intragenic deletions . Point mutations may involve the displacement of one nucleic acid base with another (base pair substitution), resulting in substitution of one amino acid for another in the final gene products, thus altering cellular function; or, 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. When a more extensive deletion occurs within a gene so that the informa- tional material of that gene is essentially lost, it is called an intragenic deletion. 14.3 EFFECT OF MUTATIONS Mutations often induce deleterious effects on the individuals or populations affected. While the effects of several individual mutagens (agents that cause muta- tions) are discussed later in this chapter, a general concept is addressed here. One of the concerns over mutagenic environmental agents is their relationship to cancer. As is widely recognized, the majority of human cancer appears to be related to environmental factors. Many mutagens have been shown to be carcinogens (cancer- causing agents) as well. However, in the long run, the ability of various environ- mental 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, indicates that 9.4 individuals 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. If a mutation occurs in such a way that a hydrophilic amino acid is substituted for a hydrophobic residue in a resultant protein, or vice versa , serious consequences can result. Sickle cell 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 approx- imately 140 amino acids in the human hemoglobin. This seemingly minor change produces abnormally shaped red blood cells that can no longer transport oxygen efficiently, leading to detrimental anemia. LA4154/frame/C14 Page 211 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC 212 ENVIRONMENTAL TOXICOLOGY On the other hand, mutations may not necessarily produce deleterious effects on an organism. For instance, if mutations occur in such a way that only one amino acid along the backbone of a protein is incorrectly specified, the three-dimensional struc- ture of the protein may not be greatly altered, allowing it to 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, but humans are highly developed organisms and when a mutation does occur, the probability is that it is a deleterious one. 14.4 INDUCTION OF MUTATION Commonly found mutagens that are of most concern to humans include UV light, ionizing radiation, microtoxins, and organic and inorganic chemicals. Some common environmental mutagens and their sources are listed in Table 14.1. 14.4.1 UV Light In the electromagnetic spectrum the region with the wavelengths from 200 to 300 nm is of primary biological importance. The main reason for this is that DNA absorbs most strongly at 260 nm. Irradiation of growth medium by UV light has been shown to cause mutations in microorganisms. 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 as a result of UV irradiation is thymine dimerization, a reaction in which two thymine molecules are fused together to form a dimer (Figure 14.3A). This dimerization may occur between adjacent thymine residues, or between two thymine residues across the chains (interchain dimerization). Dimerization results in disrup- tion of hydrogen bonding between the bases in the DNA molecule (Figure 14.3B). Chain break (P–S–P–S) is another possible result. Ultraviolet irradiation can also cause hydration of cytosine (Figure 14.4), which may also result in hydrogen bond Table 14.1 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 Chromium Metal alloys, mines Hydrazine Cigarettes and wood smoke Malonaldehyde Peroxidized polyunsaturated fatty acids Vinyl chloride Plastics Aflatoxin B 1 Fungi-contaminated grains and peanuts LA4154/frame/C14 Page 212 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC MUTAGENIC POLLUTANTS 213 disruption. The effect of UV irradiation is not limited to DNA. Proteins and RNA outside the nucleus and other cellular components can also be affected. 14.4.2 Ionizing Radiations Examples of ionizing radiations include X-rays, gamma-rays, α -particles, high- energy neutrons, and electrons. These radiations can alter DNA bases or cause fragmentation of DNA by producing single- or double-stranded breaks in phospho- Figure 14.3a UV radiation-initiated formation of a thymine dimer. Figure 14.3b Interchain dimerization disrupts hydrogen bonding between DNA bases. Figure 14.4 Hydration of cytosine. 33 6&*¤6 6&$6 33 33 6¤7$6 6776 33→ 33 6$76 6$*6 33 6¤*&6 1 1 2 1+ + ZDWHU DFLGRU KHDWLQJ XY 1 1 2 1+ + + 2+ + 2+ &\WRVLQH SKRWRSURGXFW LA4154/frame/C14 Page 213 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC 214 ENVIRONMENTAL TOXICOLOGY diester chains of the DNA molecule. In the latter case, the ions or radicals that remain along the track may cause chains of chemical reactions. 14.4.3 Chemical Mutagens As mentioned previously, approximately 70,000 commercial chemicals are in use in the United States, and this number is increasing by 1000 new compounds yearly. 2 In addition, there are many environmental chemicals that are of concern. Some of these are derived from the commercial chemicals, while others are produced from anthropogenic sources. Examples include industrial processes involving fossil fuel combustion, transportation, 3 emissions from the open burning of scrap rubber tires, combustion of agricultural wastes such as sugar cane, orchard prunings, and grain straws, municipal sewage sludges of many American cities, 4 certain herbicides such as S-(2-chloroallyl)diethyldithiocarbamate (sulfallate), 5 and textile manufac- turing processes. Mutagenic compounds have been classified into seven major categories based on their actions on DNA. The actions include alkylation, arylation, intercalation, base analog incorporation, metaphase poisons, deamination, and enzyme inhibition. 6 Table 14.2 summarizes the mechanisms involved in these categories. Some examples are given in the following sections. 14.4.3.1 Alkylating Agents Alkylating agents represent the largest group of mutagens. They may carry one, two, or more alkyl groups in a reactive form, and thus are called mono-, bi-, or polyfunctional alkylating agents. These compounds can cause base alkylation, depu- rination, backbone breakage, or alkylation of phosphate groups. For example, most nitroso compounds are highly mutagenic (and carcinogenic) because of their ability to form electrophilic species. Figure 14.5 gives an example showing how diethylni- trosamine, a nitroso compound, can act as an alkylating agent. In this case, dieth- ylnitrosamine is converted into two species, one of which is carbonium CH 3 CH 2 + 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 are highly susceptible to electrophilic attack. An alkylated guanine (G) may not base pair properly, or the information content of the molecule Table 14.2 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 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 Metaphase poisons Interference with spindle formation and disruption of migration and segregation of chromosomes LA4154/frame/C14 Page 214 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC MUTAGENIC POLLUTANTS 215 is altered in some way by the mutation. For instance, the alkylated G now pairs with T instead of pairing with C, thus causing transitional-type mutations. It is also possible that the alkyl group of N-7 labilizes the β -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. There are other mutagens whose structures are similar to one of the bases and are called base analogs . It is possible that these base analogs may be incorporated into a DNA molecule. For example, 5-bromouracil, in its normal (keto) form, hydrogen bonds with adenine (as would U or T), but in its enol form it base pairs with guanine. 14.4.3.2 Intercalating Agents Many planar aromatic hydrocarbons are thought to be able to position themselves (intercalate) between the flat layers of H-bonded base pairs in the interior of the DNA double helix, forcing it to partially uncoil. As a consequence, frame shift occurs, leading to errors in the transmission of the genetic code. These compounds are often called intercalating agents and include benzo(a)pyrene (BaP), 5-aminoacri- dine, proflavin, and others (Figure 14.6). 14.4.3.3 Metals Many studies have shown the cytotoxic effect of a variety of metallic salts on macromolecules, resulting in their denaturation. The reactions of metallic ions with Figure 14.5 Diethylnitrosoamine is an alkylating agent. Figure 14.6 Examples of intercalating agents. &+ &+ 1¤1 2&+ &+ 1¤1 2 &+ &+ _ &+ 'LHWK\OQLWURVDPLQH_ &+ & + ¤1 1¤2 7KLVVSHFLHVLVVWDELOL]HGE\UHVRQDQFH 1 1 1+ + 11+ 62 + 2 %D3 $PLQRDFULGLQH 3URIODYLQ LA4154/frame/C14 Page 215 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC 216 ENVIRONMENTAL TOXICOLOGY nucleic acids are particularly important since some of the metals can contribute to mutagenesis and carcinogenesis. The crucial factors in the toxic action of metals may involve specific reactions with certain chemical groups in biomolecules, or with certain sites in tissues or organelles. Examples were given in Chapter 12 showing the interaction of Hg and Pb with the SH group in proteins. A specific example was also presented showing the interaction of Pb with δ -aminolevulinic acid dehydratase in heme synthesis. As already noted, some toxic metals can compete with certain essential metals such as Mg, Ca, or Zn. These essential metals are required as a cofactor in certain enzyme systems or are needed to stabilize the structure of biomolecules. Research has shown that different metallic ions react with different ligands. 7 Mg 2+ and Ca 2+ ions, for example, bind to phosphate groups on nucleotides and tend to stabilize the DNA double helix, whereas Hg and Ag bind to bases and decrease the stability of the helix. Several studies have shown that chromium (Cr)(VI) compounds induce chromo- some 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 reported. 10 Cr(VI) compounds can also inhibit the activity of such enzymes as glu- tathione 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 α -tocopherol (vitamin E) was found to reduce Cr-induced chromosomal aberrations. It is thought that since vitamin E is an efficient free radical scavenger it may scavenge Cr(V) and/or free radicals. 10 14.5 REFERENCES AND SUGGESTED READINGS 1. Stryer, L., Biochemistry , 3rd ed., W. H. Freeman & Co. Publishers, San Francisco, 1988, 675. 2. Ames, B., Identifying environmental chemicals causing mutations and cancer, Sci- ence , 204, 387, 1979. 3. Pierson, W.R. et al., Mutagenicity and chemical characteristics of carbonaceous par- ticulate 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, Toxicology , 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. LA4154/frame/C14 Page 216 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC MUTAGENIC POLLUTANTS 217 10. Sugiyama, M., Lin, X., and Costa, M., Protective effect of vitamin E against chro- mosomal aberrations and mutation induced by sodium chromate in Chinese hamster V79 cells, Mutation Res ., 260, 19, 1991. 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 ., 261, 5952, 1986. 12. Shi, X. and Dalal, N.S., Chromium (V) and hydroxyl radical formation during the glutathione reductase-catalyzed reduction of chromium (VI), Biochem. Biophys. Res. Commun ., 163, 627, 1989. 14.6 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 chro- mosome 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 amino 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 mercury (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? LA4154/frame/C14 Page 217 Thursday, May 18, 2000 11:58 AM © 2001 by CRC Press LLC [...]...LA4154/frame/C14 Page 214 Thursday, May 18, 2000 11:58 AM 214 ENVIRONMENTAL TOXICOLOGY Table 14. 2 Mechanisms of Action of Several Mutagenic Agents Chemical Action Mechanism of Action Alkylation Arylation Intercalation Base analog incorporation Deamination... benzo(a)pyrene (BaP), 5-aminoacridine, proflavin, and others (Figure 14. 6) 14. 4.3.3 Metals Many studies have shown the cytotoxic effect of a variety of metallic salts on macromolecules, resulting in their denaturation The reactions of metallic ions with 1+ 1 +1 1 62 +2 1+ %D3 $PLQRDFULGLQH 3URIODYLQ Figure 14. 6 Examples of intercalating agents © 2001 by CRC Press LLC LA4154/frame/C14 Page 216 Thursday,... radicals.12 These are considered to be responsible for the observed chromate-induced DNA damage Interestingly, pretreatment with α-tocopherol (vitamin E) was found to reduce Cr-induced chromosomal aberrations It is thought that since vitamin E is an efficient free radical scavenger it may scavenge Cr(V) and/or free radicals.10 14. 5 REFERENCES AND SUGGESTED READINGS 1 Stryer, L., Biochemistry, 3rd ed.,... electrophilic species Figure 14. 5 gives an 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 CH3CH2+ 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 are... LA4154/frame/C14 Page 215 Thursday, May 18, 2000 11:58 AM MUTAGENIC POLLUTANTS &+&+ &+&+ 1¤ 1 215 &+&+ 1 ¤ 1 _ &+ _ &+ 2 'LHWK\OQLWURVDPLQH 2 &+¤ 1 1¤ 2 7KLV VSHFLHV LV VWDELOL]HG E\ UHVRQDQFH Figure 14. 5 Diethylnitrosoamine is an alkylating agent is altered in some way by the mutation For instance, the alkylated G now pairs with T instead of pairing with C, thus causing transitional-type... group of N-7 labilizes the β-glycoside bond, resulting in depurination and leading to transition or transversion Some chemical mutagens such as HNO2 can react directly with nitrogenous bases of DNA There are other mutagens whose structures are similar to one of the bases and are called base analogs It is possible that these base analogs may be incorporated into a DNA molecule For example, 5-bromouracil,... cities,4 certain herbicides such as S-(2-chloroallyl)diethyldithiocarbamate (sulfallate),5 and textile manufacturing processes Mutagenic compounds have been classified into seven major categories based on their actions on DNA The actions include alkylation, arylation, intercalation, base analog incorporation, metaphase poisons, deamination, and enzyme inhibition.6 Table 14. 2 summarizes the mechanisms involved... 14. 2 summarizes the mechanisms involved in these categories Some examples are given in the following sections 14. 4.3.1 Alkylating Agents Alkylating agents represent the largest group of mutagens They may carry one, two, 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,... case, the ions or radicals that remain along the track may cause chains of chemical reactions 14. 4.3 Chemical Mutagens As mentioned previously, approximately 70,000 commercial chemicals are in use in the United States, and this number is increasing by 1000 new compounds yearly.2 In addition, there are many environmental chemicals that are of concern Some of these are derived from the commercial chemicals,... Thursday, May 18, 2000 11:58 AM 216 ENVIRONMENTAL TOXICOLOGY nucleic acids are particularly important since some of the metals can contribute to mutagenesis and carcinogenesis The crucial factors in the toxic action of metals may involve specific reactions with certain chemical groups in biomolecules, or with certain sites in tissues or organelles Examples were given in Chapter 12 showing the interaction . cause fragmentation of DNA by producing single- or double-stranded breaks in phospho- Figure 14. 3a UV radiation-initiated formation of a thymine dimer. Figure 14. 3b Interchain dimerization disrupts. cellular components can also be affected. 14. 4.2 Ionizing Radiations Examples of ionizing radiations include X-rays, gamma-rays, α -particles, high- energy neutrons, and electrons. These. often called intercalating agents and include benzo(a)pyrene (BaP), 5-aminoacri- dine, proflavin, and others (Figure 14. 6). 14. 4.3.3 Metals Many studies have shown the cytotoxic effect of a
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