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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

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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 processdetection 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-

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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-

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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

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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

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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

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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

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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.