George Bolcsfoldi

Genetic toxicity of dopamine.

The genetic toxicity of dopamine was studied in a battery of test systems including DNA single-strand break analysis in cultured human skin fibroblasts, the Salmonella/mammalian-microsome mutagenicity test, sister-chromatid exchange analysis in human lymphocytes, the mouse-lymphoma forward mutation assay, the sex-linked recessive lethal test in Drosophila melanogaster and the micronucleus test in mouse and rat. Dopamine at concentrations of 50-300 micrograms/ml induced DNA strand breaks in human fibroblasts. It also gave a positive response in the mouse-lymphoma forward mutation assay, where a dose-dependent increase in the frequency of mutant cells was observed in the presence of dopamine, 94-750 micrograms/ml. All other tests showed no response to dopamine. The dopamine-induced DNA strand breaks in human fibroblasts were inhibited by superoxide dismutase or dithiothreitol. Furthermore, dopamine caused nicking of circular Col El DNA and bound to calf thymus DNA in vitro. It is suggested that this genetic activity of dopamine in vitro relates to oxidation of dopamine and the generation of reactive oxygen radicals, semiquinones and quinones. It is unlikely that similar reactions would occur and cause genotoxic activity of dopamine in vivo.

Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution

A rapid genotoxicity test, based on the measurement of the proportion of single- to double-stranded DNA by alkaline unwinding and hydroxyapatite elution in mouse lymphoma cells treated in vitro with various chemicals, was evaluated. Seventy-eight compounds from diverse chemical groups, including commonly tested mutagens, toxic compounds not usually tested for genotoxicity and non-toxic compounds not thought to be genotoxic were tested. The results obtained were compared with those from the mouse lymphoma TK locus forward-mutation assay, providing a basis for assessing the relative sensitivity of the 2 assays using the same cells exposed to chemicals under similar conditions. Clear evidence of DNA-damaging activity was obtained with 43 of the compounds, while 4 gave equivocal results. Of the remaining 31 compounds, 14 were toxic without inducing DNA damage while the rest were non-toxic and did not induce any DNA damage. Results were available from both the alkaline unwinding assay and the mouse lymphoma assay for 61 compounds; they showed a concordance between the 2 assays of 77%. Of the 47 compounds that were positive or equivocal in the alkaline unwinding assay, only carbon tetrachloride and prednisolone were negative in the mouse lymphoma assay, while 12 of the 19 compounds that were negative in the alkaline unwinding assay were positive in the mouse lymphoma assay. These included 3 compounds that interfere with nucleic acid metabolism, and 3 crosslinking agents, which would be expected to produce mutations to a greater extent than strand breaks. The other 6 compounds were anthranilic acid, benzoquinone, p-chloroaniline, diethylmaleate, glucose and procarbazine HCl. Of these only the last is a known carcinogen. It is concluded from the present study that there was good overall agreement between the results of the DNA alkaline unwinding and mouse lymphoma TK locus assays, but that the sensitivity of the alkaline unwinding assay is lower for some classes of compounds. Bearing this in mind, the alkaline unwinding assay is considered suitable as a rapid screen for genotoxic activity in eukaryotic cells.

Mammalian cell gene mutation assays working group report.

As part of the International Workshop on Standardization of Genotoxicity Test Procedures, in Melbourne, 27-28 February 1993, various international guidelines were examined with respect to protocol issues in the area of mammalian cell gene mutation assays. The working group on mammalian cell gene mutation assays discussed a wide range of protocol issues related to study design; in most cases the recommendations are reasonably consistent with existing guidelines. Agreement was reached on several issues as follows. The upper limit of concentration for testing non-toxic substances should be 10 mM or 5 mg/ml, whichever is lower. For testing toxic substances the criteria of an acceptable upper limit of concentration should yield 10-20% survival. Any of several established mammalian cell mutation assays (L5178Y TK+/-, CHO/HPRT, AS52/XPRT, V79/HPRT) can be used to evaluate mutagenesis in mammalian cells; the ouabain (Na/K-ATPase) system is not an acceptable mutation assay for routine evaluation of mutagenesis in mammalian cells. Ability to recover small colonies must be convincingly demonstrated when using the L5178Y TK+/- mouse lymphoma assay. In the mouse lymphoma assay (L5178Y TK+/-), colonies in positive controls and at least two (if available) representative positive doses of the test compound should be sized if a positive response is seen; in the event of a negative response due to the test compound, colony sizing of the positive control is necessary to validate the conduct of the assay. Testing both in the presence and absence of S9 metabolic activation is necessary. It was not possible to come to a firm conclusion about the length of treatment. There was a general agreement that extended treatment times (> 2 cell cycles) often bear more disadvantages than advantages and should only be used with adequate justification. It is not necessary to repeat clear positive or clear negative tests when the assay has been adequately performed; this recommendation differs significantly from the UK guidelines. If treatment groups are not replicated, the numbers of doses tested should be increased; this recommendation differs significantly from the UK guidelines. Each laboratory should establish a historical database for the performance of a given assay in that laboratory.

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay: I. In vitro sensitivity of the bacteria to 61 compounds

A differential DNA repair test was evaluated in vitro, using derivatives of E. coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+. The aim of this study was to characterize the sensitivity of the assay to different compounds in vitro and thereby provide information on the usefulness of this end-point as an indicator of genotoxicity in a host-mediated assay. Sixty-one compounds from diverse chemical groups were tested and of these 32 gave a positive result. The results obtained were compared with results from the Ames test and were in agreement for 49 out of the 61 compounds tested. Chemicals that were detected in this test but negative in the Ames test were 4-aminophenol, catechol, diethylstilbestrol, thioacetamide and thiourea. Seven of the compounds tested gave a negative result in E. coli but were positive in Salmonella. These were 4-aminobiphenyl, benzo[a]pyrene, cyclophosphamide, 1-naphthylamine, N-nitrosobutylpropylamine, quinoline and 2-toluidine. The performance of the in vitro test and reasons for the discrepant results with the Ames test are discussed. The overall concordance between the two tests was about 80%. On the basis of these results we consider these bacterial strains, and differential DNA repair as an end-point, to be sufficiently accurate as an indicator of genotoxicity in vitro and thereby also in vivo.

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay II. In vivo results for 36 compounds tested in the mouse

The aim of this study was to further evaluate the E. coli K-12 DNA repair host-mediated assay, as a short-term in vivo genotoxicity test, to be used as a complement to the micronucleus test in the routine testing of chemicals and drugs. The assay involves the administration of the test substance to mice by the route of choice, followed by the intravenous administration of a mixture of DNA repair deficient and proficient derivatives of E. coli K-12. After an incubation period the relative survival of the two strains was determined in blood, liver, lungs, kidneys and testes of the host. A significant preferential reduction of the DNA repair deficient strain in any organ indicates that the test substance possesses genotoxic properties. A total of 36 substances, 26 carcinogens, 4 weak or non-carcinogens and 6 unclassified substances, were tested in this assay. Positive results were obtained for 23 compounds. Of the carcinogens 18 were positive and of the non-carcinogens 3 were negative. The overall concordance between the assay and carcinogenicity was 72%. In general, alkylating agents and direct-acting nitroso compounds showed genotoxic activity in all organs tested, while the other substances were positive in a limited number of organs. With oral administration, which was the most commonly used administration route in the study, the organ showing a positive response most often was the blood. The results from the present study were compared with results from the micronucleus test, which were available for 26 of the substances. Results were in agreement for 15 of the substances, while 8 substances were positive in the present assay and negative in the micronucleus test: 4-aminobiphenyl, 2-anisidine, epichlorohydrin, formaldehyde, 1- and 2-naphthylamine, 2-nitrophenylenediamine and 4-nitroquinoline-N-oxide. The substances negative in the E. coli DNA repair host-mediated assay, but positive in the micronucleus test were: benzene, catechol and cyclophosphamide. It is concluded from this evaluation that the E. coli K-12 DNA repair host-mediated assay detects a number of carcinogens that are negative in the micronucleus test, while detecting most of the compounds that are positive in the latter. The advantages of this test are that differential DNA repair measures a broad spectrum of genetic damage, an in vitro/in vivo comparison is possible with the same test organisms, results can be obtained from various organs and the test is rapid.(ABSTRACT TRUNCATED AT 400 WORDS)