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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

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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

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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

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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.