Christoph Deininger

The possible role of α,β-unsaturated carbonyl compounds in mutagenesis and carcinogenesis

Abstract ga, β-Unsaturated carbonyl compounds are industrially important compounds, ubiquitous in the environment and are formed endogenously. They interact with proteins and enzymes. Genotoxicity was found in eucaryotic cells and some compounds were carcinogenic. Unsaturated carbonyl compounds are considered to play an important role in human cancer. Insufficient and contradictory results were reported on mutagenicity. We demonstrated a clear mutagenic potential for these compounds and have shown interference of their bacterial toxicity with an adequate testing. Structure-mutagenicity relationships were confirmed by the results of the SOS-chromotest. The compounds induce DNA-strand breaks. However, we did not find indications for cross linking. With mutagenic α, β-unsaturated carbonyl compounds we isolated and characterized 1, N2-cyclic deoxyguanosine adducts, 7,8-cyclic and 7-linear guanine adducts as well as 1, N27,8-biscyclic adducts and 1, N2-cyclic, 7-linear bisadducts. Reactivity of these compounds towards nucleosides runs in parallel with their mutagenic potential. Mutagenic and carcinogenic activities most probably depend on these reactions with DNA, and DNA adducts can be utilized as indicators for the role of these compounds in human carcinogenicity.

Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms: a comparison with the mutagenicity in S. typhimurium TA100

17 monofunctional methanesulphonates of widely varying structures were investigated in the SOS chromotest using the E. coli strain PQ37. All compounds tested were positive in this assay. The monofunctional methanesulphonates in general possess low SOSiP values. Five of the compounds tested i.e. iBMS, NpMS, 2 PhPMS, PkMS and 1,3-DC12PMS (for abbreviations see Table 1) did not show increasing beta-galactosidase activity and both the positive induction factors and the positive SOSiP values resulted from the toxicity correction as performed according to Quillardet and Hofnung (1985). In general methanesulphonates with a higher SN1 reactivity, in particular the secondary compounds, showed clear genotoxic activities whereas those possessing low SN1 reactivities (primary compounds) induced a low SOS repair indicating that the alkylation of O-atoms in the DNA bases contributes more to the induction of SOS repair in strain PQ37 than N-alkylations. The only exception was methyl methanesulphonate (MMS) which possessed a very high SN2 reactivity but a rather low SN1 reactivity. It had the highest SOSiP value of all tested methanesulphonates. No dependence of the genotoxicity on the SN2 reactivity could be found in this series. In general the phenyl-substituted methanesulphonates showed higher SOSiP values, which is presumably due to their relatively high SN1 reactivities and their relatively long life times in aqueous systems. There is a clear relationship between SN1 reactivities and the SOSiP values: the SOSiP values increase with rising SN1 reactivities reaching a maximum at iPMS after which the genotoxicities decrease due to the decreasing life times. The compounds with very high SN1 reactivities also possess very high hydrolysis rates. A good correlation could be established between the mutagenicities in S. typhimurium TA100 and the SOS chromotest (strain PQ37). Only 4 small deviations from this correlation could be found. The reasons for these deviations are discussed.

Genotoxicity of 1,3-dichloro-2-propanol in the SOS chromotest and in the Ames test. Elucidation of the genotoxic mechanism

1,3-Dichloro-2-propanol (1,3-DCP-OH, glycerol dichlorohydrin) is of great importance in many industrial processes and has been detected in foodstuffs, in particular in soup spices and instant soups. It has been shown to be carcinogenic, genotoxic and mutagenic. Its genotoxic mechanisms are, however, not yet entirely understood. We have investigated whether alcohol dehydrogenase (ADH) catalysed activation to the highly mutagenic and carcinogenic 1,3-dichloroacetone or formation of epichlorohydrin or other genotoxic compounds play a role for mutagenicity and genotoxicity. In our studies, no indications of ADH catalysed formation of 1,3-dichloropropane could be found, although we could demonstrate a clear activation by ADH in the case of 2-chloropropenol. Formation of allyl chloride could also be excluded. We found, however, clear evidence that epichlorohydrin formed chemically in the buffer and medium used in the test is responsible for genotoxicity. No indication was found that enzymatic formation of epichlorohydrin plays a role. Additional mutagenicity and genotoxicity studies with epichlorohydrin also confirmed the hypothesis that genotoxic effects of 1,3-DCP-OH depend on the chemical formation of epichlorohydrin.

Detection of Acrolein Congener-DNA Adducts Isolated from Cellular Systems

α,β-unsatured carbonyl compounds are potential carcinogens present in significant amounts as environmental pollutants, natural products or biological activated metabolites. This laboratory has shown that these compounds exert distinct mutagenic (Ames TA100) and genotoxic (SOS) effects. DNA adduct formation is a possible explanation in the underlying mechanism of these biological processes. Resulting from a structure reactivity study it was shown that the primary covalent binding of these compounds to the DNA bases occurs with 2’ deoxyguanosine 1 resulting in 1,N2 cyclic adducts, 2a,b or 3a,b.

Induction of sfiA SOS function by peroxides using three different E. coli strains

Five peroxides and two related compounds were tested for genotoxicity by the SOS Chromotest using 3 different E. coli strains (PQ37, PM21, GC4798). All tested hydroperoxides (hydrogen peroxide, tert-butylhydroperoxide, cumene hydroperoxide) were clearly positive in all strains. From a comparison of results obtained from the different strains it can be concluded that neither DNA lesions leading to the induction of excision repair nor covalent binding of radicals to DNA is responsible for the induction of sfiA-SOS function by hydroperoxides. Among the remaining compounds tested, only dibenzoylperoxide gave a clearly positive result in strain PQ37 whereas di-tert-butylperoxide and azobisisobutyronitrile showed only borderline activity. When using strains PM21 and GC4798, none of the latter compounds was positive. Paraquat was inactive in all strains.

Genotoxicity of 2-halosubstituted enals and 2-chloroacrylonitrile in the Ames test and the SOS-chromotest

2-Chloroacrolein and 2-bromoacrolein are very potent direct mutagens not requiring metabolic activation in Salmonella typhimurium strains TA 100 and TA 1535. Mutagenic activities decrease with increasing degree of methyl substitution at carbon atom C-3 of the acrolein moiety from 2-chloroacrolein via 2-chlorocrotonaldehyde to 2-chloro-3,3-dimethylacrolein. With 2-chloroacrylonitrile equivocal results are obtained in strain TA 100 without S9-mix and unequivocal with S9-mix. In the SOS-chromotest the 2-chloroenals are also very strong genotoxins and the structure-activity relationships found in the Ames test are clearly confirmed. 2-Chloroacrylonitrile is not positive in the SOS-chromotest. The mutagenic mechanisms are discussed, and indications are provided that genotoxicity/mutagenicity depends on formation of DNA adducts, e.g., 1,N2-cyclic deoxyguanosine adducts.

Mutagenicity of methylvinyl ketone in Salmonella typhimurium TA100 — indication for epoxidation as an activation mechanism

This paper shows that MVK is a mutagen for Salmonella typhimurium TA 100 not only in the liquid assay as previously reported, but aslo in the preincubation assay

Risk assessment for mutagenic and carcinogenic activities of α,β-unsaturated carbonyl compounds by a screening strategy based on structure-activity relationships

Abstract α,β-Unsaturated compounds are ubiquitous and are formed endogenously. They form DNA adducts and are a constant source of DNA damage. A speedy screening strategy based on structure-activity relationships and a battery of prescreening tests for a rapid and reliable assessment of the role of these compounds in mutagenesis and carcinogenesis is presented and discussed. In this screening strategy, time-consuming and expensive animal tests are replaced by in vitro test with bacteria and cell cultures. The results of the mutagenicity and genotoxicity tests, as well as the results of the binding studies of α,β-unsaturated carbonyl compounds with DNA components, and the corresponding structure-activity relationships, are presented.

Mutagenicity of 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein in Salmonella typhimurium TA 100. A comparative study

The C2-alkylated acrolein derivatives 2-methylacrolein, 2-ethylacrolein and 2-propylacrolein are mutagenic in Salmonella typhimurium TA100. They are direct mutagens, their mutagenic potency being inversely proportional to the size of the alkylating substituent in the C2 position. In the presence of S9 mix, the mutagenicity of all these substances is considerably reduced; the reduction in mutagenicity is inversely proportional to the direct mutagenic potential of the substance. As shown for 2-methylacrolein, the reduction in mutagenicity is dependent on the concentration of S9 in the S9 mix and is not significantly influenced by heat inactivation of the S9 mix or by addition of TCPO, an inhibitor of epoxide hydrolase, to the testing system. There are no indications of enzymatic activation by the metabolizing microsomal system.

Methyl methanesulphonate (MMS) is clearly mutagenic in S. typhimurium strain TA1535; a comparison with strain TA100

No mutagenicity or an uncertain mutagenic response has been reported in the literature for methyl methanesulphonate (MMS) in S. typhimurium strain TA1535 when using the plate assay. In our studies we found a reproducible mutagenic activity of 62 revertants/mumole and plate for MMS in strain TA1535 when using the preincubation assay. A dose-dependent increase in revertants was, however, observed only at fairly high doses (exceeding 4 mumole). Two different slopes were observed in the dose-response curve when testing MMS with strain TA100. Slope A is dependent on the error-prone response, possible only in strain TA100 due to the pKm101 plasmid (R factor) but not possible in strain TA1535 due to its umuDC deficiency. Slope B observed at higher doses (as in strain TA1535) could be explained through a GC----AT transition initiated by the O6-methylation of guanine. Our findings demonstrate that MMS induces back mutation in S. typhimurium strains carrying the hisG46 missense mutation due to the formation of O6-methylguanine. In the case of strain TA100 the pKm101 plasmid-mediated error-prone mechanism is, however, the predominant process in MMS mutagenesis which leads to a higher mutagenic response at much lower doses than the GT----AT transition in strain TA1535.