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

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© 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.
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© 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.


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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

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© 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.
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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

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© 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.

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© 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?

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© 2001 by CRC Press LLC