0000000000012718
AUTHOR
Georg Reifferscheid
Detection of mammalian carcinogens with an immunological DNA synthesis-inhibition test.
There is a close relationship between genotoxicity, mutagenicity and carcinogenicity. But the controversy of which short-term test system best recognizes human carcinogens is still going on. Currently, the Salmonella gene mutation assay ('Ames test') is the most widely used test for the screening of mutagens. However, many in vitro tests hold unsatisfactory validity data, presumably because of the inability of present short-term tests to detect non-genotoxic carcinogens, which are increasingly being brought into focus in the discussions of genesis of cancer. One principle often neglected in this context is the property of genotoxic agents to inhibit replicative DNA synthesis in (proliferati…
Evaluation of the SOS/umu-test post-treatment assay for the detection of genotoxic activities of pure compounds and complex environmental mixtures.
This study presents an evaluation of the SOS/umu-test after introducing an additional dilution and incubation in the post-treatment assay. This treatment reduces the influence of coloured test compounds that otherwise affect the colorimetric determination of the beta-galactosidase activity and the bacterial growth measurement during the testing of complex environmental samples. The post-treatment assay significantly increased the beta-galactosidase activity and consequently the enzyme induction ratios at higher doses of model genotoxins 4-nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, benzo(a)pyrene with low or no effect on the sensitivity of the test itsel…
Further development of the β-lactamase MutaGen assay and evaluation by comparison with Ames fluctuation tests and theumu test
A rapid, high-throughput bacterial mutagenicity test system has been developed (MutaGen test) that detects reversions of inactivating base-pair substitutions and frameshifts in a TEM-1 class A β-lactamase (ampicillinase) gene. To quickly and sensitively detect mutagens, the system utilises a series of plasmids that contain the mutated ampicillinase gene and the mucAB operon. Inactivating mutations in the ampicillinase gene include frameshifts integrated into repetitive GC-sequences and G-runs known to be mutagenic hot-spots, and base-pair substitutions inserted in or around the β-lactamase active site. Frameshift mutations completely inactivated the enzyme only when located downstream of th…
Increase of sensitivity and validity of the SOS/umu-test after replacement of the beta-galactosidase reporter gene with luciferase.
The SOS/umu-test with Salmonella typhimurium TA1535/pSK1002 as tester strain is a rapid and valuable bacterial assay for screening of umuC-dependent mutagenic potential of chemical compounds and chemicals relevant to environmental pollution. The initial assay was modified by replacing the beta-galactosidase reporter gene with luciferase. Thereby, the sensitivity of the umu-test was increased significantly and the susceptibility to intensively coloured solutions was reduced. The alternative enzyme assay in the modified umu-test (umu-Luc) represents an independent method which allows to confirm the colorimetric results obtained with the original SOS/umu-test system (umu-Gal) by measuring the …
A microplate version of the DNA-synthesis inhibition test for rapid detection of DNA-alteration potentials.
A microplate version of the DNA-synthesis inhibition test (DIT) for fast detection of DNA-alteration potentials has been developed. The DIT is based on the concept that DNA damage causes inhibition of DNA synthesis that becomes detectable some time after replicating cells have been in contact with genotoxic agents. In this test procedure human tissue culture cells (HeLa S3), prelabeled with [14C]thymidine, arfe exposed for 90 min to the substances in question. After the cells are rinsed, they are allowed to recover for 2 1/2 h in fresh culture medium, thereby unspecific interactions interfering with DNA replication are practically eliminated. Next, [3H]thymidine is added for 30 min, and the…
Genotoxicity of dental materials.
This study was performed to characterize the (possible) DNA-damaging properties of dental materials and to identify specific compounds that contribute to this genotoxicity. For screening, three tests that assay for different aspects of genotoxicity (i) the bacterial umu-test; (ii) the eucaryotic DNA synthesis inhibition test; and (iii) the in vivo alkaline filter elution technique were chosen. This investigation gives several lines of evidence that most dental materials tested (14 chemical monosubstances present in dental devices and 7 extracts of dental materials) yield 'positive' results in at least one of the genotoxicity tests, however, with effects ranging from 'borderline' to 'strong …
DNA damage susceptibility and repair in correlation to calendric age and longevity.
In two mouse strains, SAM P (senescence acceleration prone) and SAM R (senescence acceleration resistant), of different longevities, with a ratio of P/R=1:2), the DNA status in the course of aging has been investigated using the DNA Alkaline Filter Elution (AFE) technique. Six different organs (brain, liver, heart, lung, intestine, and muscle) have been used in each of the four animals of a given age. Earlier it had been shown, that DNA is damaged the more the higher the age of the animal. DNA damage susceptibility, measured after exposure of organ pieces to nitroquinoline-N-oxide (NQO), is also significantly increased at higher ages, while repair, measured of NQO damaged tissue after 3 h i…
Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay).
Abstract The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle ha…
Validation of the SOS/umu test using test results of 486 chemicals and comparison with the Ames test and carcinogenicity data
The present study gives a comprehensive update of all umu genotoxicity assay results published so far. The available data of 486 chemicals investigated with the umu test are compared with the Ames test (274 compounds) as well as rodent carcinogenicity data (179 compounds). On the whole, there is good agreement between the umu test and the Ames test results, with a concordance of about 90%. The umu test was able to detect 86% of the Ames mutagens, while the Ames test (using at least 5 strains) detected 97% of the umu positive compounds. The elimination of TA102 from the set of Ames tester strains reduced the percentage of detectable umu genotoxins from 97 to 86%. The agreement between carcin…
Molecular Composition of Glutamine Synthetase of Sinapis alba L.
Chloroplastic glutamine synthetase of Sinapis alba, purified to homogeneity by a simple three step procedure, revealed a molecular weight of about 395 kDa. The native enzyme is composed of eight subunits of identical molecular weight (about 50 kDa (each), although isoelectrofocusing yielded six distinct bands in the pH 5.6 region of the gel. Labelling of the enzyme with the glutamate analogue herbicide [14C]phosphinothricin and with [γ-32P]ATP indicated that glutamine synthetase has eight reactive centers per molecule. The native enzyme dissociated into two enzymatically active subaggregates of about 195 kDa after Mg2+ deprivation.
A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples
Abstract The umu-microtest is a miniaturized automated short-term test version proposed for screening of umuC-dependent mutagenic potentials of chemicals relevant to environmental pollution, river water and industrial waste water. The test is based on the SOS/umu-test and has been modified in order to allow extensive testing of environmental samples. Genetically engineered Salmonella typhimurium (TA1535/pSK1002) are incubated on a microplate rotor in a sloping position for 2 h with the test samples, followed by addition of fresh culture medium to reach a 10-fold dilution of the incubation medium. 2 h later, the activity of the β-galactosidase, which reflects umuC induction, is determined co…
Assessment of the mutagenic potency of sewage sludges contaminated with polycyclic aromatic hydrocarbons by an Ames sludges for fluctuation assay
9 pages, 1 figure, 6 tables.-- PMID: 14587895 [PubMed].