Dieter Wild

Bacterial β-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene

The metabolism of beta-lyase and the mutagenicity of the synthetic cysteine conjugates S-1,2-dichlorovinylcysteine (DCVC), S-1,2,2-trichlorovinylcysteine (TCVC), S-1,2,3,4,4-pentachlorobuta-1,3-dienylcysteine (PCBC) and S-3-chloropropenylcysteine (CPC) were investigated in Salmonella typhimurium strains TA100, TA2638 and TA98. The bacteria contained significantly higher concentrations of beta-lyase than mammalian subcellular fractions. Bacterial 100,000 X g supernatants cleaved benzthiazolylcysteine to equimolar amounts of mercaptobenzthiazole and pyruvate. DCVC, TCVC and PCBC produced a linear time-dependent increase in pyruvate formation when incubated with bacterial 100,000 X g supernatants; pyruvate formation was inhibited by the beta-lyase inhibitor aminooxyacetic acid (AOAA). CPC was not cleaved by bacterial enzymes to pyruvate. DCVC, TCVC and PCBC were mutagenic in three strains of S. typhimurium (TA100, TA2638 and TA98) in the Ames-test without addition of mammalian subcellular fractions; their mutagenicity was decreased by the addition of AOAA to the preincubation mixture. CPC was not mutagenic in any of the strains of bacteria tested. These results indicate that beta-lyase plays a key role in the metabolism and mutagenicity of haloalkenylcysteines when tested in S. typhimurium systems. The demonstrated formation in mammals of the mutagens DCVC, TCVC and PCBC during biotransformation of trichloroethylene (Tri), tetrachloroethylene (Tetra) and hexachlorobutadiene (HCBD) may provide a molecular explanation for the nephrocarcinogenicity of these compounds.

Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts

The use of Syrian hamster embryo cells for assessing genotoxicity provides the unique opportunity to determine 5 different end-points (gene mutations, DNA-strand breaks, aneuploidy, DNA repair (unscheduled DNA synthesis, UDS) and neoplastic transformation) in the one cell system. This approach allows direct comparisons of results produced under identical conditions of dose at target, metabolism and bioavailability. We report here on the characterization of an additional end-point in the same cell system: the formation of micronuclei indicating chromosomal changes induced by chemicals. For a preliminary validation of this new test system we have investigated 14 carcinogens and 3 non-carcinogenic structural analogues in order to evaluate the significance of micronucleus induction for carcinogenic properties. All tested carcinogens induced micronuclei in a dose-dependent manner; all non-carcinogens yielded negative results. Correlations between the formation of micronuclei and the Ames test, induction of UDS, cell transformation and the in vivo bone marrow micronucleus test are demonstrated.

Mutagenicity study of remsen-fahlberg saccharin and contaminants

Saccharin and contaminants of commercial Remsen-Fahlberg saccharin were studied for mutagenic potential with the use of the Salmonella/microsome test, Basc-test in Drosophila melanogaster and micronucleus test in mice. In none of these tests were mutagenic effects of saccharin observed. Likewise, the ortho- and para-sulfamoylbenzoic acids (OSBA and PSBA) were ineffective. Para-toluenesulfonamide (PTS) and the major contaminant ortho-toluene-sulfonamide (OTS) exhibited weak mutagenic effects in a modified Salmonella/microsome test and in Drosophila. These results do not indicate mutagenic and therewith correlated carcinogenic potential of saccharin, but they emphasize the possible activity of contaminants.

Reactivity and genotoxicity of arylnitrenium ions in bacterial and mammalian cells

Electrophilic arylnitrenium ions are considered to be the ultimate reactive intermediates formed by metabolism of mutagenic and carcinogenic arylamines and nitroarenes; they can produce DNA damage by reaction with specific sites on DNA bases. We studied their formation, reactivity and the genotoxic sequelae of their reactions with cellular DNA to understand the mutagenic and carcinogenic activities of arylamines and nitroarenes as a function of their chemical structure. Arylnitrenium ions were generated by the convenient non-metabolic procedure, photolysis of arylazides, to study the reactivity of these ultimate intermediates with DNA, by means of 32P-postlabelling, and the induction of histidine reversions in Salmonella, HPRT mutations and sister chromatid exchange in mammalian (Chinese hamster V79) cells. Good correlations were observed between the DNA-binding potencies and the mutagenic and SCE-inducing potencies of the arylnitrenium ions, among these the nitrenium ions derived from the heterocyclic food mutagens/carcinogens MeIQ, IQ, and MeIQx. This suggests that the reactivity of the arylnitrenium ions and the quantity of adducts formed with DNA are the principal determinants of the final quantity of genetic alterations in Salmonella and in V79 cells. Conversely, the quality of the adducts, that is, the structure of the arylamine residue bound, appears to be of less significance.

Chemical structure and mutagenic activity of aminoimidazoquinolines and aminonaphthimidazoles related to 2-amino-3-methylimidazo[4,5-f]quinoline.

We have synthesized 11 heterocyclic aromatic amines with chemical structures related to that of 2-amino-3-methylimidazo [4,5-f] quinoline (IQ), a potent mutagen occurring in broiled sardines, fried beef and beef extract. The mutagenic activity of these IQ analogs was studied and compared with that of IQ using the Ames test with strain TA98 of Salmonella typhimurium in presence of a metabolic activation system (S9 mix) derived from rat liver. The mutagenic activities of the IQ analogs vary over a million-fold; structure-activity comparisons indicate major contributions of the methyl substitution in the imidazole ring and of the quinoline-N, and significant contributions of methylation of the exocyclic amino group and of the geometry of the entire ring system.

Mutagenic activity in rat urine after feeding with the azo dye tartrazine

The azo dye tartrazine, after dosing by gavage, is transformed by rats into urinary metabolites which exert dose-dependent mutagenic activities in the Ames test with Salmonella typhimurium TA 98 after addition of rat liver metabolizing enzymes (S9 mix). The strain TA 100 showed no mutagenic response.

Genotoxicity study of CS (ortho-chlorobenzylidenemalononitrile) inSalmonella, Drosophila, and mice

The lacrimatory agent CS was examined for genotoxic properties. In vitro, Salmonella typhimurium was exposed to CS at concentrations up to 1.5 mg per plate and reverse mutations were assayed. In vivo: male Drosophilae were fed with CS and sex-linked recessive lethal mutations in sperm cells were assayed using the Basc test. Further, mice were exposed to CS by oral or intraperitoneal administration; bone marrow erythrocytes were analysed for chromosomal mutations by means of the micronucleus test.All experiments failed to show a mutagenic activity of CS.

Single-Electron-Transfer in the Reduction of 1, 2-Dioxetanes by Biologically Active Substrates

Substances of low oxidation potential, which can also make available protons and hydrogen atoms, e.g. phenothiazines, NADH, and ascorbic acid efficiently reduce 1,2-dioxetanes to their vic-diols by single-electron-transfer; a significant side reaction is catalytic decomposition of dioxetanes into the corresponding ketone fragments.

Genotoxicity studies of benzofuran dioxetanes and epoxides with isolated DNA, bacteria and mammalian cells.

1,2-Dioxetanes, very reactive and high energy molecules, are involved as labile intermediates in dioxygenase-activated aerobic metabolism and in physiological processes. Various toxicological tests reveal that dioxetanes are indeed genotoxic. In supercoiled DNA of bacteriophage PM2 they induce endonuclease-sensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, formamidopyrimidines). Pyrimidine dimers and sites of base loss (AP sites) which were probed by UV endonuclease and exonuclease III are minor lesions in this system. While the alkyl-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetranes such any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S. typhimurium strain TA100. DNA adducts formed with an intermediary alkylating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane. We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter, since benzofuran epoxides are highly mutagenic in the S. typhimurium strain TA100 and they form DNA adducts, as detected by the 32P-postlabelling technique. Our results imply that the type of DNA damage promoted by dioxetanes is dependent on the structural feature of dioxetanes. Furthermore, the direct photochemical DNA damage by energy transfer, i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes. Instead, photooxidation dominates in isolated DNA, while radical damage and alkylation prevail in the cellular system.

Cytogenetic effect of ortho-phenylenediamine in the mouse, Chinese hamster, and Guinea pig and of derivatives, evaluated by the micronucleus test

The mutagenic potential of ortho-phenylenediamine — known by studies on Salmonella typhimurium — was studied in mice, Chinese hamsters and Guinea pigs with the use of the micronucleus test. In bone marrow of all three species, chromosomal damage was detected following intraperitoneal injections of the test chemical, this damage was seen also after peroral administration to mice. Four derivatives of ortho-phenylenediamine containing one or two methyl-, nitro- or chlorosubstituents in 4- or 5-position of the aromatic ring were studied in mice. Of these the 4-methyl-derivative induced micronuclei, the 4-nitro-, 4,5-dimethyl-, and 4,5-dichloroderivatives were inactive. The results indicate that the chemical nature and the number of substituents influence the chromosome damaging potential.