Erwin Eder

Structure-mutagenicity relationship in α,β-unsaturated carbonylic compounds and their corresponding allylic alcohols

Abstract Several α,β-unsaturated carbonylic compounds and their corresponding allylic alcohols were tested in a modified Salmonella typhimurium mutagenicity assay according to Rannug et al. (1976) with and without metabolic activation by S9 mix. The mutagenic activities of the test substances were correlated with their chemical structures. Allyl alcohol exerted a significant direct mutagenic activity despite its lack of a good leaving (750 revertants/μmole). Glycidol, the epoxide of allyl alcohol, showed a lower direct mutagenic potential (80 revertants/μmole), whereas the corresponding aldehyde, acrolein, exhibited a direct mutagenicity 3 times higher than that of allyl alcohol. The possibility of an oxidation of allyl alcohol to its corresponding α,β-unsaturated carbonyl compound, acrolein, by bacterial alcohol dehydrogenase is discussed. The 3 methyl-substituted homologues of allyl alcohol, crotyl alcohol, 2-methallyl alcohol and 3-buten-2-ol, only showed borderline mutagenicity, while the higher homologs, 2-cyclohexen-1-ol, cinnamyl alcohol, geraniol and linalood, did not show any mutagenic properties. In contrasst with the methyl-substituted homologs of allyl alcohol, the methylated homologs of acrolein, croton aldehyde, 2-methylacrolein and methylvinyl ketone, were clearly mutagenic. Of the higher α,β-unsaturated carbonyl compounds tested in this study, only 2-cyclohexen-1-one, which has the lowest molecular weight, exerted a very small direct mutagenic effect, whereas cinnamylaldehyde and citral did not show any mutagenicity. The addition of S9 mix invariably led to a decrease in mutagenicity; this is most distinct with those compounds that had the highest direct mutagenic potential. The chemical reactivity of acrolein with deoxyadenosine was confirmed in an experiment in vitro.

Carcinogenicity of trichloroethylene: fact or artifact?

Technical trichloroethylene has been found carcinogenic in mice after high daily doses per os. A GC-MS analysis of this technical sample revealed the presence of considerable amounts of epichlorohydrin and 1.2-epoxibutane as stabilizers. These epoxides are highly mutagenic in the Ames test and are, most probably, responsible for the carcinogenic effect found in mice. The question whether trichloroethylene is carcinogenic or not remains open.ZusammenfassungTechnisches Trichloräthylen erwies sich nach hohen, täglichen oralen Dosen an Mäusen als carcinogen. Eine GC-MS-Analyse des benutzten technischen Präparates ergab die Gegenwart beträchtlicher Gehalte an Epichlorhydrin und 1.2-Epoxibutan, die im Ames-Test stark mutagen sind; diese Epoxide tragen höchstwahrscheinlich die carcinogene Wirkung in dem für den Carcinogeneseversuch verwendeten technischen Produkt, wo sie als Stabilisatoren zugesetzt werden. Die Frage, ob Trichloräthylen carcinogen ist oder nicht, bleibt offen.

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.

Correlation of alkylating and mutagenic activities of allyl and allylic compounds: standard alkylation test vs. kinetic investigation.

Thirty-nine allylic and non-allylic compounds have been tested in the standard 4-(p-nitrobenzyl)pyridine (NBP) alkylating procedure and the Salmonella typhimurium mutagenicity assay. Fourteen of these were found directly mutagenic (without addition of S-9 mix activating enzyme system). With twelve of these compounds, a good correlation of alkylating and mutagenic potencies was established; the remaining two do not meet the chemical conditions of the NBP procedure on account of HCl elimination with these two compounds. The other 25 substances were inactive in both systems. The quantitative correlation proved to be almost linear in the lower activity ranges (E approximately 2; revertants/muml approximately 600). The reasons for some deviations from the linear relationship have been analyzed and discussed on the basis of structural features. In addition to the standard alkylation test, a modified NBP-test was performed in order to obtain kinetic data and activation energy values. The results with 6 representative allylic compounds show that the overall correlation is not substantially improved above that of the standard procedure: nonetheless, additional information on reaction characteristics is obtained with some substances.

Structure-activity relationship in halogen and alkyl substituted allyl and allylic compounds: correlation of alkylating and mutagenic properties.

In a series of allylic chloroolefins and their non-allylic isomers the significance of the allylic structure and the influence of methyl and chlorine substituents on the direct mutagenic activity in Salmonella typhimurium (TA 100) was tested. The direct mutagenic potentials correlate well with the alkylating activities as measured in the nitrobenzyl-pyridine (NBP) test. In contrast to allyl chloride, the vinylic chloroolefins 2-chloro-1-propene and 1-chloro-1-propene did not show any direct mutagenic and alkylating properties. Monomethylated allylic chlorides are six to thirty times more mutagenic: 3-chloro-2-methyl-1-propene <3-chloro-1-butene <1-chloro-2-butene. The non-allylic isomers 2-chloro-2-butene and 4-chloro-1-butene, however, are not directly mutagenic. In spite of a higher alkylating potency, bimethylated allylic chlorides did not show an increase in mutagenicity if compared with monomethylated derivatives: 3-chloro-2-methyl-1-butene <1-chloro-2-methyl-2-butene. 1-Chloro-1-cyclohexene lacks mutagenic and alkylating activity, whereas 3-chloro-1-cyclohexene is comparable to allyl chloride in both respects. Dichloropropenes are also more directly mutagenic than allyl chloride: 2,3-dichloro-1-propene ⪡trans-<cis-1,3-dichloropropene. Benzyl chloride exerted the highest alkylating activity of all substances tested in this survey, and is about fifty times more mutagenic than allyl chloride. Addition of rat liver S-9 mix was followed by a distinct decrease in the mutagehicity of directly mutagenic substances, the only exception being 2,3-dichloro-1-propene, which demonstrated an increase by a factor of 35. Under the same conditions, vinylic chloroolefins are activated and become mutagenic to various degrees. Only 1-chloro-1-cyclohexene and the homoallylic compound 4-chloro-1-butene are negative both in the presence and absence of S-9 mix.

Genotoxicity and mutagenicity of the α,β-unsaturated carbonyl compound crotonaldehyde (butenal) on a plasmid shuttle vector

Crotonaldehyde is an α,β-unsaturated carbonyl compound and an important environmental and industrial toxic substance. Its mutagenic and carcinogenic properties are related to its reactivity to DNA, where it forms different guanine adducts. In order to study the mutagenic consequences of this agent in intact human cells, we treated the shuttle vector plasmid pZ189 with different doses of crotonaldehyde at 37°C for 2 h and then transfected the such damaged plasmid into the normal human lymphoblast cell line GM0621. Within these host cells the guanine adducts are repaired and the plasmids replicated by cellular enzymes. After 2.5 days replicated plasmids were purified from the cells and plasmid survival was quantitated by transformation ability. With increasing doses of crotonaldehyde, we found a significant decline of plasmid survival, reflecting a pronounced genotoxicity of crotonaldehyde-induced DNA damage in intact human cells. Using the plasmid encoded mutagenesis marker gene supF, we were able to screen for mutants and determine mutation frequency in recovered plasmids. A significant increase in mutation frequency with increasing doses of crotonaldehyde reflects mutagenicity of crotonaldehyde-induced DNA damage. Base sequence analysis of recovered mutants revealed 39% point mutations, 46% deletions, and 15% insertions and inversions. Most of the point mutations (82%) were located at G:C base pairs, which is well explained by the DNA damage profile of crotonaldehyde. Among deletions we found a frequent reoccurrence of two hot spot deletions, representing 62% of all deletions. The sites of breakpoints of these deletions hot spots and of other deletions within the plasmid were also found to be sites of DNA breaks, directly induced by crotonaldehyde, as seen in an endlabeled plasmid fragment, treated with crotonaldehyde. Further analysis of the flanking sequences around the deletion breakpoints revealed a high frequency of four different kinds of short sequence homologies of up to eight base pairs.

A new pathway of acrolein metabolism in rats

The excretion of metabolites of acrolein in rat urine after single oral administration of 10 mg/kg was investigated. S-Carboxyethylmercapturic acid (S-carboxyethyl-N-acetyl-cysteine), or S-(propionic acid methyl ester)mercapturic acid, was found as the major metabolite. An unidentified metabolite was found in expired air. Acrylic acid and methyl acrylate are suggested as intermediates in the metabolism of acrolein.

Induction of unscheduled DNA synthesis IN HeLa cells by allylic compounds

Thirteen allylic compounds, mostly with close structural relationship, were tested for their ability to induce unscheduled DNA synthesis (UDS) in HeLa cells and mutations in the Ames test; 11 induced UDS in dose dependence. Allyl isothiocyanate was negative in UDS (borderline in the Ames test) and acrolein (positive in the Ames test) proved toxic to HeLa cells, therefore UDS measurement was excluded. In general, positive qualitative and quantitative correlation between UDS, Ames test and alkylating properties (as measured in the 4-nitrobenzyl-pyridine test, NBP) were found. Among structural analogs and typical allylic compounds with various leaving groups, the amount of induced DNA repair at equimolar concentrations decreased in the same order as the mutagenic and alkylating activities in the other 2 test systems: 1,3-dichloropropene (cis) greater than 1,3-dichloropropene (trans) greater than 2,3-dichloro-1-propene; 1-chloro-2-butene greater than 3-chloro-1-butene greater than 3-chloro-2-methyl-1-propene greater than allyl chloride; allyl-methane-sulfonate greater than -iodide greater than -bromide greater than -chloride.

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.