Radioactivity mutations from

Our society has not had a very impressive record for safe disposal of industrial wastes. We have polluted our water and air, and some land areas have become virtually uninhabitable because of our improper burial of chemical wastes. As a result, many people are wary about the radioactive wastes from nuclear reactors. The potential threats of cancer and genetic mutations make these materials especially frightening. 

ADP-Ribosylation of human c-Ha-ras protein by hen liver ADP-ribosyl transferase. Human c-Ha-ras protein, normal or mutated from glycine to valine at the 12th position, was produced in Escherichia coli and purified (6). These proteins were incubated with ADP-ribosyl transferase purified from hen liver nuclei (7) in the presence of [adenylate- P]NAD. Incorporation of the radioactivity derived from p PJNAD was clearly shown at the positions corresponding to the protein bands of normal and mutated c-Ha-m.y proteins (Fig. lA and IB, lanes 3 and 4). However no significant radioactive band was formed in the absence of c-Ha-ra proteins (Fig. IB, lane 5). The incorporation of the radioactivity into c-Ha-ra.y protein was inhibited by more than 50% in the presence of 40 mM arginine methylester or 40 mM nicotinamide. 

Fig. 4. Illustration of the multiplex allele specific diagnostic assay. At the top of the panel, radioactive ohgonucleotide probes (indicated by stars) are selected by hybridization to amphcons from patient samples affixed to membrane filters. When a putative mutation-bearing allele hybridizes to a radioactive probe, it can be eluted from the filter and subjected to sequencing using chemical or radioactive dideoxy-terminator methods. This permits unequivocal identification of a large number of mutations at a high throughput.

Working with a mutated bacterial strain, Isbister et al. (62) demonstrated a novel mechanism of aerobic oxidation of dibenzothiophene which involved the specific excision of the sulfur atom from the molecule (Figure 11). Studies with -labeled dibenzothiophene showed the release of the radioactivity into the aqueous phase and ion chromatography showed the appearence of sulfate. There was no radioactive carbon dioxide released when this microorganism was incubated with 14C-labeled dibenzothiophene. GC-MS analysis showed that the oxidation product was 2,2 -dihydroxybiphenyl. Kargi and Robinson (52) also report the release of sulfate from dibenzothiophene. This OSC served as the sole carbon and sulfur source in their cultures of the aerobic thermophile Sulfolobus acidocaldarius. 

DNA molecules may be modified by the unintentional addition or deletion of nucleotides or by substituting one nucleotide for another. The result is a mutation that is transmittable to offspring. Mutations can be induced by chemical substances. This is a major concern from a toxicological viewpoint because of the detrimental effects of many mutations and because substances that cause mutations often cause cancer as well. DNA malfunction may result in birth defects, and the failure to control cell reproduction results in cancer. Radiation from x-rays and radioactivity also disrupts DNA and may cause mutation. 

DNA is available in human sperm and in white blood cells. From these sources, it should be possible to obtain DNA to detect changes resulting from agents, such as alkylating agents, that react chemically to cause mutations. In experimental animals, this can be done easily with radioactive tracers.238 Very sensitive and precise radioimmunoassays being developed may greatly improve quantitative assessment, so that this technique could be applied to experimental animals and man. No practical chemical assay has been developed that can be... 

Because uranium is a predominantly alpha-emitting radionuclide, current theories on gene mutation and chromosomal aberrations by high-LET alpha radiation suggest a potential for genotoxicity from uranium s radioactivity (BEIR 1980,1988,1990 Leach et al. 1970 Morris et al. 1990 Muller et al. 1967 Otake and Schull 1984 Sanders 1986 Stokinger et al. 1953 UNSCEAR 1982,1986,1988) (see Appendix D for a review of the hazards associated with radionuclide exposure). Other genotoxicity studies are discussed in Section 2.5. 

Fig. 1. Pertussis toxin-mediated ADP ribosylation of membrane G proteins. Isolated cell membranes (50 ng of protein) from N1E 115 cells (mouse neuroblastoma cell line), N2A cells (mouse neuroblastoma cell line), S49-1 eye cells (S49(-) mutated mouse lymphoma cell line deficient in Ga ), 549 wt cells (wild-type mouse lymphoma cell line), RBL (RBL 2H3 rat basophilic leukemia cell line), GH3 cells (GH3 rat hypophyseal tumor cell line), PC-12 (rat pheochromocytoma cell line), HIT-T15 cells (hamster insulinoma cell line), Y-1 cells (mouse adrenal cortex tumor cell line), 108 cc 15 cells (mouse/rat neuroblastoma x glioma hybrid cell line), HL-60 cells (DMSO-differentiated human leukemia cell line), HL-60 (+PT) cells (HL-60 cells pretreated with 25 ng/ml of pertussis toxin for 24 h prior to preparation of membranes), RINm5F cells (rat insulinoma cell line), and C6-2 cells (rat glioma cell line) were subjected to P-ADP-ribosylation as described in section 4.3.3. Samples were precipitated as outlined in section 4.3.5 and subjected to SDS-PAGE with separating gels containing 8% acrylamide (w/v). An autoradiogram of the dried gel is shown. Molecular masses of marker proteins are indicated (kDa). Modified Ga proteins migrate at approximately 40 kDa. Radioactivity running in front of the 30 kDa marker protein comigrates with the dye front...

The intact animal can be improved for experimental purposes if it is rendered abnormal in some way, by genetic malfunction, by illness, or by operation. Genetic defects, or mutations, are used widely in the study of bacterial metabolism, where they can be read ily induced, for example through irradiation by X-rays or from a radioactive source. Genetic defects frequently reveal themselves in the form of the absence of one specific enzyme, and metabolic studies with such enzymically defective preparations are of the same type as those made possible by the use of a specific enzymic inhibitor which we discussed above. Genetic defects in animals are rarer, but classic cases of the absence of specific enzymes and hence the accumulation of abnormal metabolites are provided in humans by the genetically carried diseases of phenylketonuria and alkaptonuria. In both, unusual substances are excreted in the urine, and the analysis of the reasons for their appearance has led to valuable information about the mechanism of amino acid metabolism in the body. 

Soon after the discoveries of X-rays and radioactivity it was learned that radiation could cause changes in matter. In 1901 P. Curie found that when a radium source was placed on his skin, woimds were produced that were difficult to heal. In 1902 skin cancer was shown to be caused by the radioactivity from radium but S years later it was learnt that radium therapy could be used to heal the disease. Large radiation doses were found to kill fungi and microorganisms and produce mutations in plants. 

Until recently, heritable changes in organisms were solely those that arose from mutations. Researchers in the field took advantage of both spontaneous mutations and those produced by exposure of organisms to radioactive materials and other substances known to induce mutations. Selective breeding was then used to increase the population of desired mutants. It was not possible to produce custom-tailored changes in genes. 

Citrullinemia is a human disease caused by a deficiency of liver arginino-succinate synthetase. It is inherited as an autosomal recessive. A fibroblast cell line from the skin of an infant with citrullinemia could not ultilize citrulline (instead of arginine) in the medium, while control normal fibroblast could (Tedesco and Mellman, 1967). For mutational studies, it may be possible either to use selective medium in which arginine is replaced with citrulline or to apply radioactive citrulline in an autoradiographic assay. 

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