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OCR for page 163
1 ~
Biologic Markers of Genetic Damage in Females
Female reproduction can be adversely
affected through a variety of mechanisms,
including damage to germ cells from pre-
natal or postnatal exposure. Resulting
damage encompasses oocyte destruction,
as well as genetic alterations that may
be transmitted to offspring. This chapter
focuses on markers of genotoxic damage
to somatic cells and to germ cells and
briefly discusses ovotoxicity. Biologic
markers are discussed under the general
headings of exposure to toxicants, oocyte
toxicity, markers of genotoxic damage or
repair, and markers of mutational events.
Direct measurements of genetic and
other germ cell damage in humans are rare,
because female germ cells are few and ac-
cess for investigation is difficult.
Work with oocytes in other mammals also
is scarce, for the same reasons. Labora-
tory and human studies have relied heavily
on reproductive end points, such as infer-
tility and fetal loss, as indicators of
germ cell damage. However, changes in
reproductive function may occur for rea-
sons unrelated to toxic exposures. There-
fore, findings from these studies may be
difficult to interpret.
Among the materials available for as-
sessing germ cell effects on female repro-
duction are gametes, other ovarian cells
and tissues, follicular fluid, cervical
and vaginal secretions, and tissue from
163
the conceptus (amnion, chorion, placenta,
embryo, and fetus). (Markers of genetic
damage in the conceptus are discussed in
Part III-Biologic Markers of Pregnancy.)
Some of these materials usually can be
obtained only through invasive procedures
and are not widely available to research-
ers. Some expendable tissues, such as
placentas and abortuses, can be used for
a few research purposes, but until recent-
ly, the only access to ovarian materials
has been through surgery (e.g., oophorec-
tomy and hysterectomy) and organ-donor
programs. In vitro fertilization and em-
bryo transfer (IVF/ET) centers present
an important research opportunity. In
some countries, investigators involved
with in vitro studies now have available
gametes, embryos, and other materials from
IVF programs. Any study based on diffi-
cult-to-obtain materials should include
a comparison with other, more widely
studied, tissues and fluids, so that the
findings of the study can be interpreted
properly.
Further systematic studies of exposed
women and their progeny that use markers
of genotoxicity (e.g., DNA adducts) or
of mutational events (e.g., micronuclei)
should improve measurement precision in
evaluating associations between exposure
and outcome. Clinical relevance of
markers should be validated. Studies
OCR for page 164
164
in New York State (Hatcher and Hook,
1981 a,b) used routine samples of biologic
material (e.g., cord blood and blood from
heel sticks) and routine birth records
to examine the relations among sister-
chromatid exchanges (SCEs), chromosomal
aberrations, and birthweight, and efforts
of this type should be extended.
MARKERS OF EXPOSURE
Biologic assessments of human genotoxic
exposure typically are accomplished
through chemical analyses or bacterial
mutagenesis assays of body fluid-usually
blood and urine, because of their availa-
bility and expendability. These tissues
can be obtained from either men or women,
so the assessments can be conducted in
both. From men, semen also can be assessed.
Recently, follicular fluid has been used
to assess exposure in females (see below).
Chemical analyses of body fluids with
gas chromatography and mass spectrophoto-
metry (GC/MS) or immunoassays give a direct
measure of exposure to specific compounds
or metabolites and the resulting internal
dose. Mutagenesis assays, however, are
indirect markers of exposure to mutagens
and are useful when exposure is unknown
or complex or when analytic standards are
lacking. Moreover, such assays demon-
strate biologic activity, rather than
simply toxicant presence.
A widely used mutagenesis assay with
potential applications in female toxicity
is the Salmonella/microsome mutagenicity
test (Ames et al., 1975), which has been
well validated and is quite sensitive to
DNA damage. It uses tester strains that
cannot synthesize the amino acid, histi-
dine, but revert and begin to grow in the
presence of mutagens. Mutagenesis assays
are generally less sensitive than such
analytic methods as GC/MS or immunoassays,
particularly for some types of compounds,
including hormones. When the mutagens
are present in a low concentration, large
sample volumes may be needed to yield
enough mutagen to be detected. Storage
and extraction procedures can distort
results, and genotoxic activity may be
compounded by body concentrations of ex-
traneous agents (e.g., foods). Nonethe-
FEAfALE REPRODUCT~ TOMCOLOGY
less, cost and performance time compare
favorably with those of chemical analyses.
New assays, such as the E. cold multitest
system (Toman et al., 1985), require small-
er biologic samples and are used to evalu-
ate several genetic and nongenetic end
points but have not been applied to human
samples.
Follicular fluid is important in oocyte
maintenance and ovulation. Because the
blood-follicle barrier is permeable,
toxicants in this milieu could affect oo-
cyte integrity, meiosis, fertilization,
and implantation. Follicular aspirates
from laparoscopies in an IVF program
have been used to measure several common
chlorinated hydrocarbons, including DOT,
PCBs, and hexachlorobenzene (Trapp et al.,
1984; Baukloh et al., 1985~. In a majority
of the 47 women sampled, most of the pol-
lutants sought were found. In these stud-
ies, the investigators noted that the oo-
cyte recovery rates and subsequent embryo
cleavage rates were inversely related to
the hydrocarbon concentrations.
MARKERS OF OOCYTE TOXICITY
Species apparently differ substantially
in oocyte sensitivity to toxicants, mole-
cular target within oocytes, and suscep-
tible stages of development. During
human fetal life, oogenesis is a sensitive
period for exposure to ovotoxicants, be-
cause germ cells damaged during this
period can not be replaced and the cells
are metabolically active. A later period
of vulnerability occurs during the preovu-
latory stage of the menstrual cycle,
when oocyte maturation resumes and the
cells are again metabolically active
(Mattison, 1982~.
Impaired fertility as a result of in
utero oocyte exposure to toxic substances
has not been proved. In mature females,
chemotherapy is associated with ovarian
failure and appears to involve damage to
growing oocytes (Chapman, 1983~. Earlier
onset of menopause in current smokers
than in nonsmokers (McKinley et al.,
1985) has been viewed as evidence that
exposures can increase oocyte atresia
rates.
Other markers of ovodepletion need to
OCR for page 165
AL4R=RS OF GENETIC DAMAGE
be developed. Two possibilities are al-
terations in gonadotropin hormones (in
that oocyte atresia is controlled by the
pituitary) and ultrasound evaluation of
ovarian size to detect gross changes. Some
of the new DNA technology may potentially
provide useful tools for detecting subtle
changes in ovarian function; for example,
ovarian DNA probes enable the measurement
of gonadal peptides (Mason et al., 1985~.
MARKERS OF GENOTOXIC DAMAGE
OR REPAIR
Much of the development and application
of genotoxic markers has been in the field
of carcinogenesis. Actual or potential
markers in humans include DNA adducts,
unscheduled DNA synthesis, and SCE. These
are not direct markers of female reproduc-
tive toxicity because, to date, they have
been used only in somatic cells. But they
hold promise as indirect markers.
DNA Adducts
DNA extracted from human tissue can be
analyzed to detect adducts formed by cova-
lent binding of a genotoxicant with a DNA
base. The significance of an adduct ap-
pears to differ according to its size,
structure, and site of binding. For ex-
ample, bulky adducts are more likely to
interfere with DNA replication than are
smaller adducts (Brusick et al., 1981~.
Studies correlating various adducts with
specific-locus mutations have indicated
that the O6-guanine adduct is critical in
mutagenesis (van Zeeland, 1986~.
Agent-specific and generic methods for
measuring adducts are available (Perera
et al., 1986; Wogan, 1988~. Agent-specific
methods permit the extent of binding of
a known agent or metabolite to be deter-
mined. Generic methods are especially
useful when the exposure or the operative
agent in the exposure is unknown (see Chap-
ters 9 and 18~.
Methods for detecting chemical-
specific adducts rely on rapid, reproduc-
ible immunoassays that use antibodies
against modified DNA, nucleotides, or
nucleosides. Binding of the antibody of
interest is measured after binding of a
1
165
radiolabeled or enzyme-linked second
antibody. These assays require approxi-
mately 300 fig of DNA (or about 300 mil of
blood) and detect adducts at a concentra-
tion of about 1 per 108 bases (Perera et al.,
1986~. The postlabeling technique (Gupta
et al., 1982) is a generic method to detect
binding by aromatic compounds that re-
quires a DNA sample of approximately 10
fig (1 ml blood) and that has a detection
level of 1 per 108-10~° bases. The assay
procedure entails digesting DNA to nucleo-
tide derivatives, labeling the nucleo-
tides with phosphorus-32, removing normal
nucleotides by thin layer chromatography,
and performing autoradiography of the 32p_
labeled nucleotides. Adducts can be char-
acterized by comparing chromatographic
patterns with those of adducts formed by
known compounds.
Assays that measure hemoglobin alkyla-
tion have been proposed as a surrogate for
immunoassays that use white-blood-cell
DNA (Ehrenberg and Osterman-Golkar,
1980~; this promising approach requires
very little blood.
DNA adducts might be measured in granulo-
sa cells found in follicular fluid or in
oocytes; no reports of this were found
in the literature on humans or experimental
animals. In the mouse, fetal adducts have
been measured to assess transplacental
DNA damage from known mutagens adminis-
tered to the mother (Lu et al., 1986~. Ad-
ducts were found in all fetal organs-gen-
erally at concentrations lower than in
maternal tissues-but the tissue distribu-
tion of adducts differed between mother
and fetus; therefore, fetal concentra-
tions could not be predicted from maternal
concentrations. In human placental tissue
(Everson et al., 1986), maternal smoking
was found to be strongly related to an ad-
duct detected by the postlabeling assay
(16 of 17 smokers compared with 3 of 14 non-
smokers). The adduct has not been charac-
terized, but appears to correlate with
the birthweight of the offspring (Everson
etal., 1988~.
DNA adducts hold promise as dosimeters
for in utero exposure to genotoxicants
that might be detrimental to developing
fetal oocytes, as well as to somatic cells.
Adducts as direct measure of germ-line
OCR for page 166
166
exposure are untested in humans. In the
laboratory, only males have been studied
for adduct formation in germinal cells,
primarily because of the inaccessibility
of female germ cells. Background concen-
trations of DNA adducts and age and sex
influences on adduct formation are sug-
gested by recent data (Randerath et al.,
1986; Reddy and Randerath, 1987~. More
information is needed to guide design and
interpretation of studies that use DNA
adducts.
Unscheduled DNA Synthesis
Measuring excision repair of adducts
as unscheduled DNA synthesis (UDS) has
been proposed as an indicator of exposure
to DNA-damaging agents (Williams, 1977~.
UDS assays require the addition of tritium-
labeled thymidine to nondividing cultured
cells. UDS is measured as the extent of
label incorporated during repair of sin-
gle-strand gaps that result from excision
of damaged nucleotides. UDS has been vali-
dated with a wide range of direct-acting
chemicals, as well as agents that require
activation (Santella, 1987~.
Oocytes appear to have effective mechan-
isms for repairing damaged DNA. UDS is a
sign of gene repair and has been observed
in oocytes of mice exposed to UV radiation
(Pedersen and Mangia, 1978~. UDS could
prove useful as a marker of reproductive
damage in humans, although no such applica-
tions have been reported.
Sister-Chromatid Exchange
SCE is the reciprocal interchange of
DNA between chromatics at one locus and
does not result in alteration of chromoso-
mal structure. SCEs reflect repair of
several types of lesions; they are more
efficiently induced by compounds that form
DNA adducts or otherwise intercalate into
DNA (e.g., alkylating agents) than by
agents that break the DNA backbone (e.g.,
radiation).
SCEs are not mutational events, nor
have they any known health consequences
(Carrano et al., 1980~. They are a manifes-
tation of repair of damaged DNA and corre-
late with specific-locus mutations in
Chinese hamster ovary cells (Carrano et
FEMALE REPRODUCTIVE TOXICOLOaY
al., 1978), but they do not correlate well
with adduct formation-not even with the
O6-guanine adduct thought to be important
in mutagenesis (Tice et al., 1984~. SCEs
are increased in patients with Bloom's
syndrome (Evans, 1982), and they generally
are accepted as indicators of potential
genetic hazards (Archer et al., 1981; Latt
etal., 1981~.
SCEs are quicker and easier to score
than chromosomal aberrations and will
often be detected even when other short-
term assays are negative (Carrano et al.,
1980~. The measurable background concen-
tration of SCEs is variable, but stand-
ardized protocols give reproducible data.
Cells are cultured from about 10 ml of
blood. Measurement of SCEs requires two
cycles of DNA replication in the presence
of bromidine deoxyuridyl, followed by
microscopic analysis of stained metaphase
cells.
SCE analysis of fetal cells has been
used to detect transplacental mutagens
in the mouse (Kram et al., 1979~; signifi-
cant increases in fetal SCEs are seen at
doses of known mutagens well below their
teratogenic doses, although fetal SCE
frequencies generally are lower than ma-
ternal frequencies (Kram et al., 1980~.
Induction of SCEs in fetal cells decreases
during gestation for direct- and indirect-
acting compounds. That may reflect reduced
placental transport, stage-dependent
influences on SCE formation~rates, or dif-
fering rates of cell turnover.
That SCEs can be induced by such repro-
ductive toxins as antineoplastic drugs
(Norppa et al., 1980) and ethylene oxide
(Yager et al., 1983) indicates their util-
ity as exposure markers. Conflicting find-
ings suggest that SCEs are uncertain in-
dicators of in utero exposure or markers
of fetal damage.
MARKERS OF MUTATIONAL
EVENTS
In considering mutational events, chro-
mosomal lesions and point mutations are
of interest. Because of the inaccessibil-
ity of female germ cells, work to date has
relied heavily on observations in other
species, mainly the mouse.
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MARKERS OF GENETIC DAMAGE
Laboratory Animals
The work in rodent systems is described
in detail in Chapter 8 (see also Russell
and Shelby, 1985~. Tests for genetic dam-
age are conducted less often in female
than in male animals, because the yield
of mutations is lower. The dominant-leth-
al, heritable-translocation, and specif-
ic-locus tests are the most extensively
used laboratory methods; in all of these,
risk is evaluated in Fat progeny. The domi-
nant-lethal test measures early embryonic
death induced by a variety of chromosomal
abnormalities, primarily numerical, rath-
er than structural (Brusick, 1980~. The
heritable-translocation assay measures
transmissible structural chromosomal
lesions, and the specific-locus test can
measure recessive point mutations. Dobson
and Felton ~ 1983) have underscored the
importance of accounting for species dif-
ferences in target and cell killing when
extrapolating genetic risk in laboratory
animals to human females.
A protocol for cytogenetic analysis of
meiosis II oocytes and first-cleavage
zygotes recently was proposed as a sensi-
tive assay for aneuploidy in meiosis I and
II cells, as well as for structural aberra-
tions after DNA synthesis (Mailhes et al.,
1986~. A measure of aneuploidy in female
germ cells is of great interest because
more than 80% of aneuploidy in humans ori-
ginates there (Hassold, 1985~.
Human Oocytes
Although analysis of human oocyte chro-
mosomes has been attempted with material
from ovariectomies or biopsies (Jagiello
and Lin, 1974; Jagiello et al., 1975),
early cytogenetic studies of meiotic cells
were hampered by technical difficulties
in chromosome preparation. More recently,
new chromosome preparation techniques
have been applied to develop estimates
of chromosomal aberrations with oocytes
from stimulated cycles of infertile women
(Wramsby et al., 1987~. The resulting data
place the background frequency of oocyte
chromosomal anomalies at 50%, but the es-
timate is limited by the select nature and
small size of the population studied and
167
by the unknown effect of ovulation induc-
tion. However, those limitations do not
preclude using IVF materials to examine
cytogenetic characteristics of the egg
in relation to successful fertilization
and implantation. In addition, it should
be possible to compare results in infertile
women with those obtained with oocytes
from unstimulated cycles of fertile women.
Successful efforts also have been made
to analyze chromosomal anomalies in early
human embryos obtained from IVF (Angell
etal., 1983~.
Human Somatic Cells
Alterations in chromosomal structure
are unequivocal markers of genetic dam-
age. The basic lesion thought to under-
lie all structural anomalies is a break
in the chromatin fiber. Aberrations in
somatic cells are considered undesirable,
but not directly predictive of health ef-
fects. Chromosomal aberrations are gener-
ally less sensitive measures of chemical
exposure, which tend to induce aberrations
only when the cell is in the S-phase (i.e.,
chromatic aberration detectable as SCEs)
(Morgan and Wolff, 1984~. In addition,
scoring aberrations can be time-consum-
~ng.
Significant increases in spontaneous
abortions or heritable chromosomal abnor-
malities have not been found among
Japanese who survived the atomic bomb
blasts, despite somatic cell chromosomal
damage (Schull et al., 1981 a); however,
the observed trends of increasing frequen-
cy with increasing dose received were pre-
dicted (see also discussion in the Chapter
9~. An early study of chromosomal aberra-
tions in newborns suggested an association
with low birthweight (Bochkov, 1974), but
the relationship was not confirmed in a
later study that used matched, contempora-
neous controls (Hatcher and Hook, 1981 a).
Micronuclei
Micronuclei are microscopically
visible DNA-containing bodies in the cyto-
plasm of a cell with no structural connec-
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168
tion to the nucleus. The presence of these
extranuclear bodies is considered to indi-
cate chromosomal damage (aneuploidy, as
well as breakage). Metaphase analysis
is not required, so the test is relatively
easy and inexpensive, and it is amenable
to automation. The background rate of
micronuclei is very low (less than 1%),
so many cells are required for analysis.
A major limitation of this test is that
the human spleen eventually removes
blood cells with micronuclei. Early ap-
plications of the micronucleus test used
exfoliated surface cells. but more recent
v ,
assessments used bone marrow cells.
which can be obtained only through invasive
procedures. Efforts are under way to apply
the method to more accessible cell popula-
tions, such as peripheral lymphocytes.
A technique is needed to pick up cells at
the first mitoses; a proposed method uses
an inhibitor to prevent cytoplasmic divi-
sion after nuclear division (Fenech and
Morley, 1986~.
The micronucleus test has been used in
rodents with fetal liver erythroblasts
as a model system for transplacentally
induced chromosomal damage. Because ro-
dent fetal liver is metabolically more
active than adult bone marrow, the trans-
placental test is generally more sensitive
to genotoxic agents (Cole et al., 1983),
as well as being better suited for assess-
ing risks to the fetus from maternal ex-
posures (King and Wild, 1979~. No reports
of analogous applications of micronuclei
in humans were found.
FEMALE REPRODUCTIVE TOXICOLOGY
Specific-Locus Mutations
Standardized tests to detect specific-
locus mutations in somatic cells are only
beginning to be available. Mutation rates
may be lower in germ cells than in somatic
cells, because of differences in repair
rates. (In addition, female germ cells
undergo fewer doublings than male germ
cells.) Nonetheless, these tests are like-
ly to prove useful, especially to elucidate
differences in species.
The hypoxanthine phosphoribosyl trans-
ferase (HPRT) specific-locus test was one
of the first to detect spontaneously oc-
curring mutants in cultured human lympho-
cYtes. In this test, mutants are detected
as cells resistant to azaguanine or thio-
ouanine in the culture medium. A high fre-
quency of thioguanine-resistant cells
has been reported in patients treated with
known mutagens (Archer et al., 1981~.
Recently, a test for glycophorin A
loss was applied to red blood cells of Jap-
anese atomic bomb survivors; mutations
were linearly related to the estimated
radiation dose (Langlois et al., 1987~.
Jensen and Thilly (1986) reported that
characteristic mutation spectra are pro-
duced in human B lymphocytes by specific
chemicals, and gradient denaturing gel
electrophoresis might be usable for gener-
ating a mutation spectrum from as little
as 10 ml of blood (Liber et al., 1985~.
The tests mentioned here are relatively
new and have not been broadly applied.
Representative terms from entire chapter:
dna adducts
