National Academies Press: OpenBook

Biologic Markers in Reproductive Toxicology (1989)

Chapter: 18. Molecular Biology: Developing DNA Markers of Genotoxic Effects

« Previous: 17. Introduction
Suggested Citation:"18. Molecular Biology: Developing DNA Markers of Genotoxic Effects." National Research Council. 1989. Biologic Markers in Reproductive Toxicology. Washington, DC: The National Academies Press. doi: 10.17226/774.
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Page 211
Suggested Citation:"18. Molecular Biology: Developing DNA Markers of Genotoxic Effects." National Research Council. 1989. Biologic Markers in Reproductive Toxicology. Washington, DC: The National Academies Press. doi: 10.17226/774.
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Page 212
Suggested Citation:"18. Molecular Biology: Developing DNA Markers of Genotoxic Effects." National Research Council. 1989. Biologic Markers in Reproductive Toxicology. Washington, DC: The National Academies Press. doi: 10.17226/774.
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Page 213
Suggested Citation:"18. Molecular Biology: Developing DNA Markers of Genotoxic Effects." National Research Council. 1989. Biologic Markers in Reproductive Toxicology. Washington, DC: The National Academies Press. doi: 10.17226/774.
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Page 214

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18 Molecular Biology: Developing DNA Markers of Genolox~c Effects This chapter briefly discusses the ef- fect of molecular biology on prenatal diag- nosis. The assays are discussed in detail in Chapters 9 and 12. The ability to obtain DNA from the fetus has been possible for the past 10 years through amniocentesis after 16 weeks of gestation. However, techniques to sample chorion in the first trimester and to kary- otype the sample directly without long- term tissue culture recently became pos- sible. The chorionic biopsy consists of aspiration of chorionic villi through the cervical canal or transabdominally with ultrasound guidance. Results of chromoso- mal analysis are available within days, rather than the weeks required with conven- tional techniques. Without the need for cultured preparations, direct karyotyping could prove amenable to analysis of chromo- somal aberrations possibly associated with early loss (1-6 weeks after implanta- tion). Also, DNA is readily available from the collected tissue or from cultured cells derived from the sampling. DETECTING HERITABLE GENETIC DAMAGE One of the most interesting innovations in prenatal diagnosis is the use of DNA probes that reveal genetic markers near 211 specific genes (McDonough, 1985) (Table 18-1~. DNA probes have been applied for prenatal diagnosis of cystic fibrosis, and predictive testing for the gene for Huntington's disease began. In addition, the gene for Duchenne muscular dystrophy has been sequenced and the so-called "re- cessive oncogene" responsible for famili- al predisposition to retinoblastoma was discovered. The number of probes available is increasing exponentially. A genetic marker is a segment of DNA that lies near a unidentified gene that is involved in the disease etiology. With DNA probes and genetic markers, it might be possible to detect most of the more than 3,000 conditions caused by single-gene mutations. Applications of molecular biology to clinical medicine will change the approach to diagnosis (McDonough, 1985~. Molecular diagnosis—even during prenatal life— is possible with two related techniques (see Chapter 12 for details): · Restriction - fragment- length poly- morphisms. Highly specific restriction endonucleases cut DNA between particular base sequences. When altered by mutation, DNA is severed into fragments of a size different from normal. The homozygous and heterozygous states can be differen-

212 TABLE 1~1 Disorders Diagnosable by Analysis of Cellular DNA Validated Uses of DNA Analysis for Diagnosis Sickl~cell anemia B-thalessemia x-thalessem~a Factor VIII deficiency Factor IX deficiency Phenylketonuna a~-antitrypsin deficiency Hunungton's disease Antithromb~n III deficiency Orn~thine transcarbamylase deficiency Duchenne muscular dystrophy Argninosucc~n~c acid dehydrogenase deficiency Osteogenesis imperfects type II Congenital adrenal hyperplasia Probable Uses of DNA Analysis for Diagnosis Fragile X syndrome Adult-onset poll ystic kidney disease Source: McDonough, 1985. tiated by comparing abnormal fragment size with normal fragment size. Potentially, these can be used even if the gene leading to the disease state is unknown. Abnor- malities of the genes for hemoglobin (whose deficiency results in sickle-cell ane- mia), growth hormone, and 21-hydroxy- lase (whose deficiency results in congeni- tal adrenal hyperplasia) are conditions that can be diagnosed with this approach. Also, these have been used to diagnose several other conditions, including Huntington's disease, phenylketonuria, factor VIII and factor IX deficiencies, and §-thalassemia. · Oligonucleotide probes. When the precise DNA mutation is known, but the mutation cannot be discriminated with a restriction-enzyme cut, oligonucleotide probes representing the normal and abnor- mal sequences can be used to identify the genotypes. As increasing numbers of normal and mutated gene sequences become identified, the practicality of this tech- nique will increase. DNA technology is versatile. Every mono- genic disorder potentially is diagnosable with DNA probes, as increasing numbers of probes and polymorphisms are recognized and restriction enzymes are developed. TOXICI7YDURING PREGNANCY The explication of the molecular map of the human genome will be accompanied by the development of functional correla- tions that might provide insights into the basic pathogenesis of most disorders, including those caused by chemical muta- gens and physical factors (such as radia- tion). Within the next decade, many dis- eases and toxic conditions probably will be defined in molecular terms and become subject to diagnosis from a few microliters of blood (Ward et al., 1983~. DNA probes might be used to detect chemical or food contaminants in the body. Tests based on such DNA probes could replace current as- says, because of their greater sensitivity and speed. Quality control may suffer as DNA probes are used more widely, particularly if commercial kits become available. Ac- curacy is essential, and reliability could be diminished because of such prob- lems as incomplete DNA digestion, faulty hybridization, contamination, and mis- labeling. Even in the best of hands, inter- pretation of these tests and associated family counseling require extensive ex- perience and commitment. In addition, the validity of the tests in the absence of confirmatory assays is a problem. A1- though revolutionary developments in DNA probes have enormous potential as biologic markers in prenatal diagnosis, many chal- lenges lie ahead. MARKERS OF EXPOSURE With the rapid development of monoclonal antibodies, radioimmunoassays, and mole- cular genetic technologies, new tech- niques have been developed to detect toxi- cants covalently bound to DNA to form adducts (Wogan and Gorelick, 1985; Perera, 1986; Wogan, 1988~. Many chemicals that are active carcinogens or mutagens either are electrophilic or are converted to elec- trophilic metabolites. These may become bound to DNA, RNA, orproteins. The conse- quences of these adducts have not been clearly demonstrated, but they are thought to initiate carcinogenesis or mutagenesis (Wogan and Gorelick, 1985~. DNA adducts have been measured in blood. Cord blood has also been used (Daffos et

MARKERS OF GENOTOXIC EFFECTS al., 1985; Reddy and Randerath, 1988~. Theoretically, it should be possible to use amniotic cells or chorionic villus cells to determine the fetal exposure to genotoxic chemicals. The amount of tissue required by the assays is large for these sampling procedures. But improvements in the laboratory procedures might make the assays possible on smaller samples. In humans, assessment of in utero expo- sure to DNA-damaging agents has been at- tempted by comparing SCE frequencies in blood from mothers and their offspring. A case report from Sweden described in- creased SCEs in four children of two labor- atory technicians who worked during preg- nancy (Funes-Cravioto et al., 1977~. Ar- dito et al. (1980) compared SCE frequencies in smoking and nonsmoking mothers and their infants' cord blood and found that mean 213 SCE frequency was slightly higher in moth- ers than in the newborns. No difference was found between frequencies in maternal or cord blood of smokers and nonsmokers. In a similar study of smoking mothers and alcoholic mothers (Seshadri et al., 1982), the SCE frequency only in drinking mothers was higher than that in controls ( 13.5 versus 10.95 SCE/cell), but the SCE rate in their infants was not significantly increased (9.71 versus 8.95 SCE/cell). In a separate analysis, neonates with neur- al tube defects were found to have higher rates of SCEs than normal babies ( 10.34 versus 8.95 SCE/cell) (Seshadri et al., 1982~. A systematic study of infants with normal and reduced birthweights found no association of growth retardation with SCEs measured in cord and postnatal blood (Hatcher and Hook, 1981 b).

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Does exposure to environmental toxicants inhibit our ability to have healthy children who develop normally? Biologic markers—indicators that can tell us when environmental factors have caused a change at the cellular or biochemical level that might affect reproductive ability—are a promising tool for research aimed at answering that important question. Biologic Markers in Reproductive Toxicology examines the potential of these markers in environmental health studies; clarifies definitions, underlying concepts, and possible applications; and shows the benefits to be gained from their use in reproductive and neurodevelopmental research.

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