Biologic Markers in Reproductive Toxicology (1989) / Chapter Skim
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9. Markers for Measuring Germinal Genetic Toxicity and Heritable Mutations in People
9. Markers for Measuring Germinal Genetic Toxicity and Heritable Mutations in People
Pages 119-140
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From page 119... ...
Thus, accidental, occupational, environmental, or therapeutic exposures to ionizing radiation or some chemicals might cause germinal mutations that will reduce a personts ability to produce normal, healthy children. When dealing with animal data, the evaluation of genetic toxicity involves the detection of genetic damage, measurement of genetic effects, and extrapolation of results.
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From page 120... ...
The development of markers of genetic damage in the human line is proceeding in three interrelated directions. The directions involve research and development leading to markers of exposure of human MILE REPRODUCTIVE TOXlCOLo~ germ cells to genotoxic agents, to markers of germinal mutations (i.e., mutations in the germ cells of mutagen-exposed persons)
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From page 121... ...
As illustrated in Figure 9-1, ger- and germinal mutations and for detecting minal mutations induced early in game- genetic damage in human male germ cells. - ' This chapter reviews the available methods for measuring human heritable mutations, briefly discussing the findings of studies with mutagen-exposed populations.
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From page 122... ...
Exposure of parent to genotoxin Germinal mutations in stem cells or differentiating germ cells B Mutations detected in the offspring of mutagenized parents Heritable mutations at birth Mitosis, Mitosis differentiation differentiation S Heritable mutations ~ ~ mutations et > during development ', first cleavage ~ S division M4LE REPRODUCTIVE TOXICOLOGY Mitosis, meiosis, differentiation Germinal mutations In mature gametes _' Fertilization _' FIGURE 9-1 Schematic representation of human genuline, including some of the cell types In which genetic damage can be measured.
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From page 123... ...
. Fetuses with numerical chromosomal abnormalities are at increased risk of being aborted spontaneously; numerical abnormalities contribute to a major part of spontaneous abortions and stillbirths.
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From page 124... ...
of the true incidence of cytogenetic abnormalities in germ cells or fertilized eggs, analyses of newborns show a range of frequencies of 2 in 100,000 for inversions to 121 in 100,000 for trisomy 21, the most common numerical chromosomal abnormality at birth (Table 9-3~. These data are based on analyses of MALE REPRODUCTIVE TOXICOLOGY 67,014 offspring from six countries (Sankaranarayanan, 1982~.
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From page 125... ...
However, it says little about the mutability of the noncoding part of human DNA and is limited to DNA changes that affect protein electrophoretic mobility. It also is labor-intensive and requires large populations for the detection of even moderate effects that might be due to mutagen exposure.
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From page 126... ...
In the future, these sequences might be used to develop nucleic acid probes to locate and characMALE REPRODUCTIVE TOXICOLOGY terize the corresponding DNA in the human genome. RESULTS OF EPIDEMIOLOGIC STUDIES OF HUMAN HERITABLE MUTATIONS IN EXPOSED POPULATIONS Atomic Bomb Survivors The three heritable-mutation-detecting methods noted above (sentinel phenotypes, chromosomal aberrations, and mutant proteins)
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From page 127... ...
The most accessible human tissue is blood, and all the somatic tests under development have used red or white blood cells. Instead of examining DNA directly, they are designed to detect rare cells with marker phenotypes from among large numbers of mostly normal cells through the use of selective growth conditions or mechanical methods.
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From page 128... ...
Research being conducted with this method is aimed at characterizing the sources of measurement variation, applying it to people exposed to and not MALE REPRODUCTIVE TOXICOLOGY exposed to mutagens, and investigating the molecular nature of the mutations. Detection of Somatic Mutations in Red Blood Cells Unlike white blood cells, mature red blood cells contain neither nuclei nor DNA.
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From page 129... ...
Later developments have made it possible to cut genomic DNA into small pieces with restriction enzymes and then to use chemical methods to determine the nucleotide sequences within each fragment (Church and Gilbert, 1984~. Although these methods are in common use, a recent estimate determined that only 4 x 106 total base pairs had been sequenced by this technique-the equivalent of slightly more than 0.001 of the human haploid genome.
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From page 130... ...
FIGURE 9-2 Production of restnction-length polymorphisms using restriction enzymes and separation of different DNA fragment sizes by Agarose gel electrophoresis. Source: OTA, 1986.
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From page 131... ...
(my) Separate DNA fragments using agarose gel electrophoresis.
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From page 132... ...
Alternative approaches that retain the idea of comparing parental genomes with those of offspring by hybridization have been proposed and may yet prove feasible. Pulse-Field Gel Electrophoresis When DNA is cut with restriction enzymes and run in gel electrophoresis, it forms so many bands that it appears as a smear of DNA.
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From page 133... ...
Two-Dimensional Denaturing Gradient Gel Electrophoresis Lerman ( 1985) proposed an additional technique that uses two-dimensional electrophoretic separation of child and parental DNA to discriminate three types of DNA: sequences common to all three, polymorphisms that are inherited from one parent, and sequences with new mutations.
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From page 134... ...
Using restriction enzymes, cut DNA into (double-stranded) fragments of various lengths.
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From page 135... ...
In spite of the technical difficulties, it is expected that these human cytogenetic data bases will continue to increase in size and will gain importance in establishing spontaneous rates of chromosomal abnormalities in the germ line of the human male. The most convincing, and in fact the only direct, evidence of the sensitivity of human germ cells to agent-induced genetic damage has been the demonstration by Brewen et al.
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From page 136... ...
Sperm methods obviously are limited to males; to date, there are no direct methods that can be applied to assess genetic damage in the germ cells of females. As stated earlier, damage inherited via female germ cells must be assessed by survey or by analysis of mutations using offspring methods.
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From page 137... ...
The markers designed to be quantitative markers suitable for estimating chromosomal and genie mutation frequencies in germ cells include the markers of cytogenetic abnormalities, gene mutations, and aneuploidy in sperm. As discussed below, none of these methods has been sufficiently developed or validated for use in large-scale studies of people exposed to mutagens.
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From page 138... ...
DNA Adduction in Sperm Many chemicals that break chromosomes and induce gene mutations do so via DNA adduction that interferes with DNA replication and repair. Specific DNA adducts have been measured in the testicular germ cells and sperm of mutagen-exposed mice (Sega and Owens, 1983)
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From page 139... ...
In principle, these probes provide a means of detecting sperm that are aneuploid for sex chromosomes or autosomes (Joseph et al., 1984; Wyrobek and Pinkel, 1986~. Applied to somatic and germ cells of the same person, this technique could provide a means for comparing induction and persistence of chromosomal aneuploidy in somatic and germinal cells of mutagen-exposed men.
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From page 140... ...
However, as emphasized in this chapter, the available methods for detecting human germline mutations are inefficient and inadequate for most human exposures. Future research should emphasize and be aimed toward the promising new cellular and molecular approaches using semen and offspring tissue (Table 9-4, column 4~.
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