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OCR for page 107
8
Assessing Transmitted Mutations in Mice
This chapter reviews laboratory tests
for assessing exposure or heritable genet-
ic effects of exposure in laboratory ani-
mals. Genetic damage can occur in somatic
cells and in germ cells. Induced genetic
damage in germ cells can lead to altera-
tions in cell functions or cell death.
Alternatively, induced genetic damage
can be transmitted to the next generation,
in which case the conceptus might suffer
no ill effects or might have undesirable
manifestations (including death) during
some or all stages of life (prenatal and
postnatal).
Mutagenic chemicals and radiation of
various kinds are widely distributed in
the environment. The mutagens that attract
the most attention are products of the
chemical industry, but some exist natural-
ly in the environment (NRC, 1982~. Many
genetic systems are available for mutagen-
icity screening. For the purpose of this
report, only end points of mutational dam-
age to mammalian germ cells will be con-
sidered. When a chemical to which humans
are exposed causes mutations in a labora-
tory mammal, such as the mouse, the genetic
risk associated with human exposure to
the chemical becomes a matter of serious
concern. During the last decade, many
chemicals under development for possible
use in or as drugs, cosmetics, and food
additives have undergone mutagenicity
107
testing. A dilemma arises when a chemical,
either under development or already in
the human environment, is of value to at
least some segments of society and has
been shown to be mutagenic. It is difficult
to determine the largest human exposure
that poses no substantial harm to human
health. Determination of such an exposure
is referred to as genetic risk assessment.
Some environmental chemicals cause
genetic damage to germ cells of experimen-
tal mammals-e."., ethylene oxide, tri-
methyl phosphate, dibromochloropropane
(DBCP), acrylamide, bisacrylamide, and
many cancer chemotherapeutic drugs. It
is assumed that these chemicals will be
mutagenic lo human germ cells under appro-
priate conditions. Direct study of chemi-
cally induced transmitted genetic effects
(mutations) in humans is virtually impos-
sible, so the risk must be estimated from
a variety of experimental test systems.
The systems are usually categorized into
two groups-mammalian germline (MG) and
nonmammalian germline (NMG). Several
ways of evaluating genetic risk have been
proposed; they differ not only in how MG
data are used, but also in the emphasis
placed on NMG data.
There is no consensus on how to assess
genetic risk associated with environmen-
tal chemical mutagens, and acceptable
strategy and guidelines are crucially
OCR for page 108
108
needed (NRC, 1982, 1983~. In the United
States, no chemical has ever been regulated
on the basis of its potential for increas-
ing the mutation load of later generations,
nor has genetic risk evaluation contrib-
uted to the regulatory decision-making
process (OTA, 1986~. Regardless of the
specifics accepted for genetic risk as-
sessment, data on transmissible genetic
effects in laboratory mammals will be in-
dispensable-not only as a measure of end
points, but also to form a standard for
evaluating the usefulness of results of
NMG tests as indicators of genetic risk
to humans.
Assessment of the genetic risk associ-
ated with exposure to a chemical includes
several components:
· Defensible evidence that the chemical
in question has the potential to induce
genetic damage to human germ cells.
· Identification and quantification
in experimental systems of the types of
mutations that are expected to be produced
and transmitted to the next generation.
· Extrapolation of experimental results
to humans (i.e., quantification of the
increase expected for each class of muta-
tion associated with likely human expo-
sures).
· Estimation of the expected total con-
tribution to the human genetic load.
· Estimation of the impact of the expect-
ed mutational increase on society.
The list is formidable. If progress is to
be made in practical genetic risk assess-
ment, a simple concept that makes use of
carefully selected biologic markers
needs to be adopted.
Evaluation of genetic risk of a chemical
follows a three-step process—detection
of mutagenicity, measurement of genetic
effects, and extrapolation of results.
Chemical mutagens can vary in the manner
in which they react with various cellular
and chromosomal components. Conse-
quently, the genetic damage that they pro-
duce can vary, and no test system can meas-
ure every conceivable type of genetic
damage. Methods for measuring some of the
end points have been established; methods
for measuring others are still under de-
AL'9LE REPRODUCTIVE TOXICOLOGY
velopment. Obviously, it is impractical,
as well as expensive, to use all the estab-
lished tests for transmissible genetic
effects for every chemical that needs
to be evaluated. Therefore, a simple con-
cept must be developed for the purpose of
practical genetic risk assessment.
ASSESSING MARKERS IN
LABORATORY ANIMALS
Our understanding of the mechanism
and effects of interaction between xeno-
biotic substances and mammalian DNA
comes predominantly from in vitro and
in viva studies of animal cells. The sus-
ceptibility of the male parent to induced
mutations in reproductive cells was demon-
strated by Muller with irradiated Drosoph-
ila males 6 decades ago (Muller, 1927~.
Since the 1940s, laboratory mice have been
intensively studied for spontaneous and
induced gene mutations and chromosomal
abnormalities. Radiation and over half
the approximately 20 chemicals tested so
far in mice induced heritable mutations
in mouse male germ cells (as measured by
the specific-locus-mutation and herit-
able-translocation tests). As yet, there
is no validated murine test for measuring
chemically induced germline mutations
directly in the germ cells of exposed
males, and all germinal mutagenicity in
mice is inferred from heritable-mutagen-
icity tests. Generally, there has been
good agreement between results in somatic
cells in viva and heritable effects of
treated differentiating male germ cells.
However, the induction of somatic muta-
tions has not been predictive of mutagenic-
ity in spermatogenic stem cells. Only a
subset of agents that induce mutations
in somatic cells also induces mutations
in the spermatogenic stem cells. Continu-
ing studies in mice have attempted to re-
veal the nature of the selective immutabil-
ity of stem cells by some agents, and their
results have suggested that these cells
have high repair capability. Studies in
mice might also be used to investigate the
molecular aspects of the different somatic
and germinal mutational responses. This
work might identify specific genetic le-
sions for which there is high somatic-ger-
OCR for page 109
TRANSM17TED ~lJTATIONS IN MICE
minal concordance. Studies in mice have
been and will continue to be the corner-
stone of our understanding of the basic
aspects of spontaneous and induced muta-
tions. And, the mouse will continue to play
a key role in the quest to understand the
molecular nature of mutations and their
effect on phenotype and health.
An accepted tenet of toxicology and car-
cinogen testing is that we should not
rely on one species for risk extrapolations
to human beings. It is well known that me-
tabolic activation is required for the
toxicity, carcinogenicity, and mutagenic-
ity of some chemicals and that animals and
people can differ in their metah~li.sm
For example, the germinal effects of ex-
posure to DBCP differ markedly among spe-
cies (Wyrobek et al., in press). It kills
spermatogenic cells in most species, in-
cluding rats, hamsters, rabbits, and hu-
mans; and it induces dominant lethal muta-
tions in treated male rats and might induce
spontaneous abortions in the spouses of
exposed men. Mice are the only animals
known whose male germ cells are essentially
nonresponsive to DBCP, showing neither
toxic nor mutational effects. Thus, for
DBCP, the mouse would be a poor choice as
a test species for estimating human ger-
minal toxicity and mutational risks.
Mouse-human discrepancies have been ob-
served also with the germinal toxicity
of some cancer chemotherapeutic agents,
such as adriamycin (Meistrich et al.,
1985~. Possible solutions for the problem
associated with interspecies extrapola-
tion would be the use of molecular dosime-
try (e.g., DNA or protein adducts) to de-
velop quantitative methods for extrapola-
tion, the development of a second labora-
tory species for measuring germinal and
heritable mutations, and the development
of methods for detecting germinal and her-
itable mutations directly in people.
MARKERS OF EXPOSURE
Some measure of exposure is necessary,
not only to establish whether a chemical
or its active metabolite reached the germ
cells, but also to relate a genetic re-
sponse to specific molecular target
sites qualitatively and quantitatively.
109
When the genetic response is clearly posi-
tive, the question of whether the chemical
reached the target cell is academic. Ab-
sence of a genetic response can mean that
the chemical did not reach the germ cells,
that the chemical is a nonmutagen, or that
the chemical is a mutagen but the test sys-
tem is insensitive or has an inherently
effective repair capability. Thus, mark-
ers of exposure are necessary for proper
interpretation of results.
The markers that indicate exposure of
male and female germ cells were discussed
in detail recently by Russell and Shelby
(1985~. They can be classified into the
following categories: cytotoxicity, cy-
togenetic effects, cellular biochemical
responses, molecular binding, and
cellular morphologic responses.
Cytotoxicity
Cytotoxicity to some germ cells implies
that the test chemical reached the gonads
and supports the assumption that the sur-
viving cells were also exposed. Cytotoxic
effects might be determined directly by
histologically examining the seminiferous
tubules (or of the ovary for female ani-
mals) at an appropriate interval (usually
days) after exposure, allowing for the
manifestation of cellular degeneration
or for the disappearance of affected cells.
When specific germ cell stages are scored
separately, the method is sensitive. Very
low levels of cell-killing that might not
result in a demonstrable effect on fertili-
ty might be detected. Often, reproductive
performance can be affected; without his-
tologic verification, however, that is
unreliable as a measure of germ cell ex-
posure, because fertility can also be
reduced by nongerminal means.
Cytogenetic Effects
Demonstrable chromosomal damage is
direct evidence of exposure. The cytoge-
netic end points used widely are chromoso-
mal aberration, sister-chromatic exchange
(SCE), and micronucleus formation. In
all cases, scoring is done in descendants
of exposed cells. In males, chromosomal
aberrations can be scored in spermatogoni-
OCR for page 110
110
al metaphases, in meiocytes, and in the
zygotic metaphase; micronucleus informa-
tion in spermatogonia, in spermatids, and
in two-cell embryos; and SCE in spermato-
gonial and meiotic metaphases. In females,
chromosomal aberrations can be scored in
the metaphase-II and in the zygotic meta-
phase stages and micronucleus in two-cell
embryos. (SCE induction in female germ
cells has not been reported.) SCEs can
occur in the presence or absence of demon-
strable chromosomal aberrations and point
mutations. Micronucleus formation is
generally believed to result from the
chromosomal elimination that follows
chromosomal breakage or misdivision.
Biochemical Responses
Introduction of exogenous substances
into the cell elicits enzymatic responses.
In the case of mutagens, DNA damage (an
indication of exposure) can trigger un-
scheduled DNA synthesis (UDS) in some
male and female germ cell stages. Sper-
matocytes, spermatids, and oocytes do not
normally undergo DNA synthesis; however,
when chemicals bind to DNA, these germ
cells respond by repairing some altered
sites. If the germ cells are provided with
radioactive thymidine during repair, the
amount of repair activity (thus, the amount
of DNA damage) can be measured.
Molecular Binding
One of the most direct measures of germ
cell exposure is the demonstration of mo-
lecular binding. In the context of muta-
genesis, the most important molecular
target sites are the chromosomal DNA and
proteins (histones and protamines). Vari-
ous techniques of molecular dosimetry
can be used to measure very low frequencies
of adduct formation through the reaction
of the test chemical or its metabolite with
germ cell DNA. If the germ cell stage stud-
ied in molecular dosimetry and in mutagene-
sis is the same, it is possible to relate
the magnitude and quality of DNA binding
to mutation induction. This is the most
sensitive marker of exposure so far, al-
though adduct formation in germ cells has
been studied only in males.
MALE REPRODUCTIVE TOXICOLOGY
Cellular Morphologic Responses
During spermatogenesis, cells undergo
changes that culminate in spermatozoa with
the morphologic characteristics of their
species (see previous chapters for extend-
ed discussion). Changes in sperm structure
that result from chemical exposure of the
male indicate toxicity either directly
to the maturing spermatogenic cells or
indirectly through damage to other sys-
tems. The distinction between the two
types of toxic response is difficult to
make.
TESTS IN MICE TO DETERMINE
TRANSMITTED GENETIC EFFECTS
Chemical mutagens react with various
cellular and chromosomal components in
different ways, so they produce different
types of genetic damage. Generally, muta-
tions are of two types: either gene (or
point) mutations and small deficiencies
or chromosomal aberrations (changes in
chromosomal structure or number). The
tests of induced transmitted mutations
in mice are summarized in Table 8-1. The
genetic tests that are involved with these
markers have been discussed in detail by
Russell and Shelby (1985~; what follows
here is a brief summary.
Specific-Locus Test with Visible
Markers
This is the most widely used system for
detecting induced point mutations and
small deficiencies. The test makes use
of genetic information on up to seven loci
that affect visible characteristics of
the animal. Animals of one strain, which
has normal (or wild-type) alleles at all
seven loci, are exposed to the test agent
and then mated with animals of a tester
stock, which is homozygous for recessive
alleles at all the loci. Normally, all
the progeny would resemble the wild-type
parent. However, if mutations are induced
at any of the loci, the type or distribution
of visible characteristics-such as coat
pigment, eye color, hair structure, or
structure of the external ear-might be
affected. The test can be used to study
OCR for page 111
111
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OCR for page 112
112
mutational response in both male and female
germ cells.
Specific-Locus Test with Biochemical
Markers
Induced point mutations and small defi-
ciencies can change protein structure
and thus alter the electrophoretic mobil-
ity of proteins or, in the case of enzymes,
alter the magnitude of enzyme activity.
Electrophoresis and enzyme activity as-
sessments have therefore been used on a
limited scale to screen for induced bio-
chemical mutations in male germ cells.
No study in females has been reported. A
heritable altered electrophoretic pattern
is a good marker for mutation in the struc-
tural gene, but alteration of enzyme ac-
tivity can also arise as a result of genetic
change at sites other than the structural
gene. Because there are many more biochem-
ical markers than visible markers, these
tests have the advantage that they are more
likely to detect a toxic effect. However,
the biochemical methods have the distinct
disadvantage that tissues need to be re-
moved by biopsy or processed from blood.
Electrophoresis and enzyme activity as-
sessments have been developed and used
widely, but improvement in tissue process-
ing and electrophoretic techniques can
be expected. For additional references
on these methods see Feuers and Bishop
(1986), Lewis and Johnson (1986), J. Peters
et al. ( 1986), and Pretsch ( 1986~.
Specific-Locus Test with Immunologic
Markers (H Test)
This test is based on the large number
of H genes that control cell surface anti-
gens that induce histocompatibility re-
sponses. There are about 40 H genes in
the mouse. Mutations might result in a new
antigenic form or the loss of an antigen.
Detection of the mutants is based on skin-
graft rejection patterns in a transplanta-
tion scheme that involves exposed mice
and tester strains. The test detects pri-
marily intragenic changes and small defi-
ciencies in the H loci. It has been used
only in males. The use of the test has been
restricted because transplantation of
ABLE REPRODUCTIVE TOXICOLOGY
skin grafts is a surgical procedure whose
outcome can be influenced by nongenetic
factors.
Recessive Lethal Test
Induced mutations in this class are leth-
al in the homozygous or hemizygous state,
that is, when both copies of the gene are
mutant alleles or when the single copy
of the sex-linked gene in males is in the
mutant form. Most of these lethal muta-
tions are small deficiencies and small
intragenic changes. However, induced
reciprocal translocations that are lethal
in homozygous conditions are observed
occasionally; in these situations, a
translocation of genetic material from
one chromosome to another alters the ex-
pression or structure of the gene in such
a way that the condition is lethal in the
homozygous animal.
Two methods for detecting the small muta-
tional changes have been attempted or pro-
posed. In one method, the entire genome
is screened. That requires three succes-
sive generations of specific mating pat-
terns. Daughters in the final generation
are mated to their sires. The presence of
mutations is detected as an increase in
intrauterine death rate due to homozygosi-
ty for the mutations among the conceptuses.
In the other proposed method, only specific
chromosomal segments, either autosomes
or X chromosomes, are screened; this makes
use of inversions with genetic markers.
The second method requires two or three
successive generations of specific mating
patterns. If a recessive lethal mutation
is present in the inverted segment or is
closely linked to it, resulting concep-
tuses that are homozygous for the inverted
segment containing the mutation have an
increased risk of fetal death. Therefore,
these genetically marked progeny are eith-
er absent or reduced in number in the final
generation.
Dominant Lethal Test
Dominant lethal mutations cause death
among first-generation progeny. General-
ly, death occurs either during an early
cleavage stage (in which case the affected
OCR for page 113
TRANSMITTED MUTATIONS IN MICE
embryo fails to implant) or around the time
of implantation (in which case the affected
embryo stimulates decidual reactions and
the formation of a resorption body in
mice). Dominant lethal mutations reflect
primarily chromosomal breaks. Embryonic
lethality occurs because of the resulting
deletions and asymmetric exchanges.
The dominant lethal test has been the
most widely used in viva mammalian mutagen-
icity test and has been used to study genet-
ic effects in male and female germ cells.
Increased embryonic lethality after ex-
posure of male parents indicates clasto-
genicity (that is, induced chromosomal
breakage) in male germ cells. However,
increased embryonic lethality after ex-
posure of female parents can result from
genetic or nongenetic causes (that is,
uterine damage). The effect might reflect
chromosomal breakage or a phenomenon
acting through the maternal environment.
Observation of chromosomal aberrations
in the pronuclear metaphase or of micronu-
clei in two-cell embryos would strongly
favor genetic causation.
Effects of dominant lethal mutations
can also be measured with an in vitro tech-
nique. The biologic marker in this case
is the ability of two-cell embrvn.~ tn rap_
velop to the trophectoderm outgrowth
stage when cultured in vitro. The relative
frequency of successful development of
embryos of the experimental group, com-
pared with the control group, is used to
determine the dominant lethal mutation
rate. This in vitro procedure allows dis-
tinction between Reimplantation loss
due to dominant lethal mutations and
reduced implantation due to reduced
fertilization.
Heritable-Translocation Test
Reciprocal exchange of genetic material
between nonhomologous chromosomes is much
more readily inducible in male than in
female germ cells. With chemicals, there-
fore, this test has been used solely in
studies involving exposed males. When
a reciprocal exchange is induced in a male
germ cell, the resulting progeny are heter-
ozygous for the translocation.
113
The two methods used to screen for trans-
location carriers are both based on meiotic
and segregation properties of the hetero-
zygous offspring. The first method is
fertility testing for semisterility
(i.e., reduced number of living concep-
tuses). Semisterility is expressed when
translocation-heterozygous offspring
are mated to normal animals. From these
matings, slightly more than half (on the
average) of conceptuses produced have
unbalanced chromosomal constitutions;
that is, about half the fetuses have dupli-
cation and deficiency in the region that
has undergone translocations. Inasmuch
as some types of exchanges cause blockage
in early spermatogenesis, this method also
screens for completely sterile transloca-
tion carriers. Chromosomes of meiotic
or somatic cells can be analyzed for veri-
fication of translocations. The other
method is direct cytologic examination
of meiotic cells for multivalent chromoso-
mal association; that is, the translocated
chromosomes will form quadrivalent, in-
stead of the normal bivalent, associa-
tions. Breeding tests in females often
require an extra generation, and cytoge-
netic analysis of oocytes is hampered by
the limitation in the number of oocytes
that can be analyzed and by the relative
complexity of the procedure. Therefore,
screening is restricted to progeny of ex-
posed males.
Inversions
Inversions are chromosomal rearrange-
ments that involve a segment within one
chromosome. The segment's orientation
of the transcription process is inverted.
Because all chromosomes in the standard
mouse are telocentric in nature (that is,
the centromere is near the end of a chromo-
some), inversions are paracentric (do not
include the centromere). Detection of
progeny carrying newly induced inversion
is done cytologically. Crossing-over
within the inversion produces a dicentric
chromatic, which results in the formation
of a bridge in the first anaphase. This
cytologic test is more easily applied to
male, than to female, progeny. The use of
inversions as a biologic marker of induced
OCR for page 114
114
chromosomal breakage and rearrangement
is not likely to be important because of
the low induction rate and the cytologic
scoring procedure, which requires
technical expertise and is time consuming.
Sex-Chromosome Loss
In the mouse, the XO condition (only
one X chromosome and no other sex chromo-
some) results in viable females, and the
YO condition (a Y chromosome and no other
sex chromosome) is lethal. Theoretically,
these conditions arise either through
chromosomal breakage and elimination or
through nondisjunction (improper separa-
tion of chromosome pairs). So far, how-
ever, the induced XO condition is only the
result of chromosomal breakage. Detection
of mutation is based on the differential
expression of X-linked markers in hemizy-
gous (XO) and heterozygous (XX) female
progeny. The XO condition is verified
cytologically or by breeding tests. Sex-
chromosome loss has been screened for in
offspring of male and female exposed
parents, although only to a limited extent.
Nondisjunction Test
Nondisjunction leads to unequal dis-
tribution of homologous chromosomes in
progeny cells; that is, a progeny cell
will contain either too many or too few
chromosomes. In the standard mouse, when
autosomes are involved, the animal dies.
Autosomal monosomic animals (i.e., with
only one copy of a particular autosome)
die early in embryonic development, where-
as autosomal trisomic animals (i.e.,
with three copies of a particular autosome)
might survive up to late fetal and early
postnatal stages. Except for animals
with the YO condition, all other monosomic
and trisomic products of sex-chromosomal
nondisjunction are viable. Because mono-
somy can also be produced via chromosom-
al breakage, trisomies are the most reli-
able biologic markers of transmitted
nondisjunctional products.
Cytologic evidence of nondisjunction
induced in germ cells has been reported,
but there is no clear evidence of a trans-
mitted induced aneuploidy. Trisomy of
AL4LE REPRODUCTIVE TOXICOLOGY
the sex chromosomes appears to be the
most promising biologic marker in experi-
mental aneuploidy, because these off-
spring are viable. Trisomies among
progeny of exposed parents can be detected
either visually or by reproductive tests.
The former method makes use of X-linked
markers that determine visible charac-
teristics; the latter is based on the find-
ing that XXY and XYY males are sterile.
In both cases, aneuploidy can be verified
cytologically.
One proposed method of testing for in-
ducible nondisjunction uses genetic mark-
ers on autosomes in high-nondisjunction
tester stock. The high-nondisjunction
tester stock produces a high frequency
of gametes that either lack the marked
chromosome (nullisomic) or have an extra
copy of the marked chromosome (disomic).
If the mutagenic treatment of the exposed
mice produces nullisomy and disomy of the
same chromosome, the complementing com-
binations from the matings of the exposed
mice and the tester stock (i.e., pairing
of nullisomic and disomic gametes) should
result in viable conceptuses. Some of the
nondisjunction progeny are detected on
the basis of external genetic markers ex-
pressed in viable complementing types.
This method is complex, and it is not clear
how useful it will be in large-scale
testing.
Aneuploidy can be scored in zygotic pro-
nuclear metaphases. At this stage of the
conceptus, the male and female contribu-
tions are still separate, and often they
are distinguishable from one another.
False-positive test results might occur,
because loss of chromosomes can result
from the cytologic procedure. Therefore,
a more reliable indicator of aneuploidy
is the presence of at least one extra chro-
mosome; i.e., the presence of an extra
chromosome is a better criterion than the
presence of too few chromosomes. However,
the reliability of the cytologic data re-
mains doubtful unless they are matched
with similar findings in later embryonic
and postnatal stages.
One method worth exploring is the late-
fetal-death method. In mice and rats, dead
implants are expressed primarily as re-
sorption bodies. Midgestation and late
OCR for page 115
TRANSM17TED HllTATIONS IN MICE
fetal death are uncommon. Most autosomal
trisomies cause lethality during the sec-
ond half of gestation, so induced trisomies
can be scored by uterine examination in
late gestation. For additional refer-
ences, see Russell (1985) and Searle and
Beechey (1985~.
Cytogenetic Analysis of Zygotes
The pronuclear metaphase stage has
been analyzed for numerical and structural
chromosomal anomalies after exposure of
male and female germ cells to an agent be-
fore, at, or after fertilization. Struc-
tural aberrations that can be scored in-
clude deletions, exchanges, and chromoso-
mal fragmentation. This method is useful
in followup studies of suspected chromoso-
mal effects that lead to embryonic mortali-
ty (dominant lethals), particularly when
the exposed parents are female.
Tests for Dominant Mutations
Mutations that have dominant or semi-
dominant effects are the most important
class of mutations for the next generation,
because the mutations will affect about
half of these progeny. Dominant mutations
vary from small intragenic changes to gross
chromosomal exchanges. Several methods
have been used to detect mutagen-induced
increases in dominant mutations. The mark-
ers of first-generation effects that have
been used to date are also varied, affect-
ing either specific organs, organ systems,
or function or any visually detected un-
usual phenotype. They are miscellaneous
phenotypes detected postnatally, congen-
ital anomalies in fetuses, abnormal
enzyme activities, sperm anomalies, be-
havioral changes, cataract development,
and skeletal changes. The last two have
been the most useful for risk evaluation,
because the bases for measuring mutation
rates and risk are the best established.
Further improvement in methods of meas-
uring rates of induction of dominant mu-
tations is needed. Common among these
methods are the problems associated with
incomplete penetrance, variable expres-
sivity, and expression of variant pheno-
type caused by nongenetic factors. As more
115
data become available, a well-defined set
of phenotypic markers and loci might be
developed for mutant detection and count-
ing. The skeletal and cataract systems
are progressing in this direction.
NEEDED RESEARCH ON GENETIC
DAMAGE IN LABORATORY
ANIMALS
Base changes, DNA deletions, gene trans-
position through chromosomal rearrange-
ments, and chromosomal misdivision are
the genetic changes generally recognized
as the major mechanisms of induced mutagen-
esis. Integration of transposable ele-
ments to new sites is also emerging as a
mechanism.
Male and female germ cells and the vari-
ous germ cell stages differ in many ways,
including ability to repair DNA lesions,
length of cell-cycle time, and interval
between S phases. In somatic cell systems,
each cell is autonomous with respect to
the fixation of aberrations. In the case
of male meiotic and postmeiotic germ cells,
however, the fixation of chromosomal
breaks and exchanges is a joint venture
between the fertilizing sperm and the egg.
The sperm brings in the premutational le-
sion, and the fertilized egg either repairs
it or processes it into a break and ex-
change. Chemical mutagens can differ from
one another in the degree to which each
chromosomal target site reacts, but no
known mutagen binds to only a single molec-
ular entity. Finally, mutagens bind not
only to DNA sites, but also to chromosomal
and extrachromosomal proteins. Taken
together, all these factors exemplify the
complexities involved in understanding
the mechanisms, from the initial step of
the mutation process (adduct formation)
to the expression of mutation in con-
centuses. We still have only a minimal
understanding of these mechanisms.
Relationship Between Molecular Target
Sites and Production of Various Types of
Mutations
To understand this relationship, one
must keep in mind not only the reaction
properties and molecular nature of the
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116
mutation, but also the biologic properties
of various germ cell stages. Thus, in addi-
tion to studies of the relation between
the different adducts formed with germ
cell chromosomal DNA and protein and the
types of transmitted genetic effects pro-
duced, studies of repair of specific DNA
adducts are also essential. It is general-
ly assumed that base adducts are the impor-
tant reaction products in DNA. But the
oxygen of the phosphate backbone (forming
phosphotriesters) is also a target, and
it is the primary site of alkylation for
some alkylating mutagens, such as iso-
propyl methane sulfonate and ethylnitro-
sourea. The questions of whether phospho-
triesters have mutational consequence
and whether mammalian germ cells have cor-
responding specific repair enzymes await
detailed studies.
Alkylation of protamines has been hy-
pothesized to lead to chromosomal break-
age. The extent to which the hypothesis
is true needs to be investigated further.
Molecular Nature and Expression of
Mutations
Transmitted genetic damage that has
dominant or semidominant expression is
especially important in genetic risk con-
siderations, because it usually shows
up in the first generation. This class of
mutation includes both gene mutations and
chromosomal rearrangement. The molecular
nature of mutagen-induced genetic damage,
the way in which deleterious effects are
expressed, and why some mutations have
incomplete penetrance or highly variable
expression are important problems-not
only for risk considerations, but also
for basic genetics. With the rapid devel-
opment of DNA experimentation, solutions
of these problems are now accessible.
One clue to the molecular nature of ge-
netic damage might come from reciprocal
translocations. The question has been
raised whether some human genetic disor-
ders that have been assumed to result
from single gene mutations could instead
be associated with chromosomal rearrange-
ments. It has been generally believed that
balanced reciprocal translocations do
not involve loss or gain in chromosomal
MALE REPRODUCTIVE TOMCOLOGY
components. However, anincreasing number
of clear associations between balanced
exchange and deleterious effects suggest
that the breakpoint might be in a struc-
tural gene or that it might affect the ac-
tivity of genes in the immediate vicinity.
Stocks of mice are available for DNA se-
quencing and for gene-expression studies.
More than 400 sites of autosomal dominant
mutations in the human genome are recog-
nized; many of them involve serious disor-
ders. Most human genetic disorders fail
to yield simple Mendelian ratios, and these
disorders are sometimes referred to as
irregularly inherited. Results of
studies of induced dominant skeletal muta-
tions in mice suggest that many irregularly
inherited disorders might also result from
single dominant mutations with incomplete
penetrance. Furthermore, these mutations
have pleiotropic effects when they are
expressed. Stocks of mice are available
for studying the types of DNA damage that
cause dominant skeletal defects and how
the mutations influence development to
cause variability in expression and pleio-
tropic manifestations.
Mutagen-Caused Induction of
Integration of Endogenous Transposable
Elements in New Sites
That the insertion of transposable
gene elements in the vicinity of, or onto,
any given gene might cause a variant pheno-
typic expression of the gene-originally
suggested by Barbara McClintock in the
late 1940s-is now a well-established ge-
neticphenomenon. Forexample,manyspon-
taneous mutations in the white locus of
Drosophila species are caused by the inser-
tion of transposons, such as the copia
element; the dilute locus of laboratory
mice is associated with the insertion of
an ecotropic murine leukemia virus (MuLV)
~enome: and virus-induced oncogenesis
involves the insertion of retroviral regu-
latory gene elements at a proto-oncogene
locus (converting it to an oncogene).
Although it is not yet known, it is general-
ly believed that there are many endogenous
transposable elements in the mammalian
genome. If exposure of germ cells to muta-
gens can induce integration of endogenous
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TRANSMITTED MUTATIONS IN MICE
transposable elements in new sites, the
fundamental and risk implications are
great. The issue must be resolved through
exhaustive studies.
Mouse Mutants as Models of Human
Genetic Diseases
Many laboratories in the United States
have rich collections of spontaneously
occurring or mutagen-induced mouse muta-
tions. Many of these mutations are useful
in studying the development of genetic
disorders that are similar to those found
in humans. Three examples of modeling are
described here.
Deficiency in the enzyme ornithine car-
bamoyltransferase is known in human and
mouse mutants and results in urinary de-
fects. The mouse sparse-fur mutant has,
besides its hair abnormality, the tendency
to produce kidney or bladder stones that
are composed primarily of erotic acid.
The sparse-fur locus is on the X chromo-
some, and a quantitative measure of X inac-
tivation can be studied by examining the
amount of enzyme present in animals in
which the X chromosome has been fragmented
in translocation mutants. The location
of the presumptive X inactivation center
on the X chromosome might thus be deter-
mined. A biologic marker in this case
causes a physiologic defect that is used
as a tool to study the basic problem of the
natural inactivation of one of the two X
chromosomes.
An electrophoretic variant of the
enzyme pyruvate kinase known in the mouse
results from an alteration of the gene on
chromosome 9. This locus occurs in the
region where many chromosomal deletions
have been isolated. By determining
whether the enzyme variant is present in
F. progeny of crosses that involve the dele-
tion mutant and the pyruvate kinase elec-
trophoretic mutant, one can further
define the boundary of the deletion. The
human pyruvate kinase can also be distin-
guished from the mouse enzyme electropho-
retically; following this enzyme in mouse-
human hybrid cells makes it possible to
identify the chromosomal location. The
biologic marker in this case is useful for
genetic mapping studies.
117
Synthesis of the neurotransmitters
serotonin and norepinephrine and conver-
sion of phenylalanine to tyrosine are car-
ried out by enzymes that all use a common
cofactor, tetrahydrobiopterin (BH4~.
The biosynthesis of BH4 proceeds from guan-
osine triphosphate through a pathway that
involves three or four enzymes. Human and
mouse mutants that result in a reduced
concentration of BH4 have recently been
identified. In the human case, a mental
deficiency, atypical phenylketonuria
is the phenotypic expression of the muta-
tion; in the mouse, several behavioral
abnormalities are manifested. The biolog-
ic markers that need to be examined are the
individual enzymes, so that the regulation
and normal function of BH4 can be understood
and means of alleviating the defects in
such mutants can be devised.
Nondisjunction
Aneuploidy resulting from chromosomal
missegregation constitutes an important
fraction of transmitted human genetic
anomalies. The extent to which it is in-
ducible by chemicals in male or female
cells is not clear, mainly because no chem-
ical has been clearly established as an
inducer of nondisjunction in these cells.
Conceivably, chromosomal missegregation
results from damage to the spindle and
kinetochore and their precursors or via
chromosomal rearrangement, which, in
turn, could affect normal pairing and seg-
regation of homologous chromosomes during
meiotic stages. For the latter possible
mechanism, it is essential to know when
synapsis actually takes place during sper-
matogenesis. A provocative hypothesis
stating that synapsis and recombination
occur during the last premeiotic 5 phase,
rather than later during zygotene and pach-
ytene, respectively, has been raised.
The issue needs to be resolved.
DNA Methods
Failure to detect increases in mutation
rates in supposedly mutagenized human
populations has triggered interest in the
use of molecular methods that entail
direct analysis of human DNA. A recent
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118
publication of a workshop report (Dele-
hanty et al., 1986) identified six DNA
methods to detect human heritable muta-
tions (see also Chapter 9~. It cautioned,
however, that none of the methods was ready
for field application but that refinements
in DNA experimentation, would soon permit
the analysis of mutation in human popula-
tions. Because of the dynamic nature of
DNA experimentation, it is assumed that
better methods will eventually become
available for use in human genetic epidemi-
ology.
The report (Delehanty et al., 1986) enu-
merated several properties as essential
for new methods to be successful. They must
be able to examine 10~° base pairs; detect
MALE REPRODUCTIVE TOXICOLOGY
a well-defined and wide spectrum of muta-
tional end points; have extremely low error
rates; use easily accessible samples;
conserve time, people, and resources; cope
with the complexity of the human genome;
and recognize recombination, polymorph-
ism, physically variable genes, somatic
mutations masquerading as heritable muta-
tions, and false paternity. Those proper-
ties also constitute one of the main rea-
sons why appropriate mutagenesis studies
in laboratory mice are necessary. Research
in mice with chemical mutagens would not
only contribute to method development,
but also provide the basis for interpreting
human results.
Representative terms from entire chapter:
genetic damage
