Michael Scheutwinkel-Reich

Bacterial mutagenicity of the tranquilizing constituents of Valerianaceae roots

The valepotriates valtrate/isovaltrate and dihydrovaltrate are considered to be the main tranquilizing constituents of drugs derived from the roots of several Valerianaceae. The decomposition products of valtrate and isovaltrate include the metabolites baldrinal and homobaldrinal, respectively, whereas the decomposition products of dihydrovaltrate do not include baldrinal-like metabolites. Purified valtrate/isovaltrate, dihydrovaltrate, baldrinal and homobaldrinal were investigated for their genotoxic activity in the Salmonella/microsome test and the SOS-chromotest. The valepotriates developed mutagenic activity in these test systems only in the presence of S9 mix, whereas both baldrinals showed mutagenic effects in both tests with and without metabolic activation.

Microbiological studies investigating mutagenicity of deep frying fat fractions and some of their components

In this study, the Salmonella/microsome mutagenicity test according to Ames et al. (Mutation Res. 31∶347, 1975) was performed in order to detect possible mutagenicity of oxidized deep frying fat fractions. Furthermore, the mono-, di-, tri- and tetrahydroxyoctadecanoic acids and the hydroperoxide of linoleic acid were investigated as model test substances. The Ames assay was carried out with and without metabolic activation including preincubation and liquid culture procedures as described by Mitchell (Mutation Res. 54∶1, 1978). The results show no mutagenic effects for the oxidized fractions of deep frying fats nor for the model test substances. At higher concentrations, however, limited test reliability resulted from direct toxic effects on bacterial growth.

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase locus, but not in the Na+/K+ ATPase locus in V79 hamster cells

The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase (HPRT) locus as measured by 6-thioguanine resistance (TGr) in V79 hamster cells. Alu I does not induce mutations in the Na+/K+ ATPase locus as measured by ouabain resistance (OUAr). The data are interpreted to mean that most if not all Alu I-induced TGr mutations represent chromosomal aberrations.

Detection of hydroxy fatty acids in biological samples using capillary gas chromatography in combination with positive and negative chemical ionization mass spectrometry

The most common method for use in the structural analysis of hydroxy fatty acids in biological samples is the gas chromatography-mass spectrometry (GC-MS) analysis of trimethylsilyl ethers of the methyl esters using electron impact ionization. A comparison of electron impact (EI) with chemical ionization mass spectrometry (CI-MS) shows that CI-MS is the superior technique. All ions necessary for structural analysis are observed at sufficiently high levels of intensity when methane or isobutane are used as reactant gases. The molecular weight can be determined from the ion group M+H, M-15 and M+H−90. The ionic series M+H−n×90 enables one to determine the number of hydroxyl groups. The position of the hydroxyl groups can be derived from the fragments of the α-cleavage of the fatty acid chain. The application of heptafluorobutyrates as derivatives for hydroxy fatty acid methyl esters shows advantages in the trace analysis of these compounds. Heptafluorobutyrates exhibit useful mass fragmentation patterns in the positive as well as in the negative CI mode. With methane as the reactant gas, M+H usually is base peak in positive mass spectra. The ionic series M+H−n×214 leads to the number of hydroxy groups in the molecule. In the negative mass spectra, M and M-20 are indicative for the molecular weight. The ion group m/z 213, 194 and 178 at high levels of intensity is typical for heptafluorobutyrates. The advantage of the application of heptafluorobutyrates is the high sensitivity which can be obtained in trace analysis using negative MS. Heptafluorobutyrates of hydroxy fatty acids gave a 20-fold higher response in the negative scan mode compared to that of the positive. The detection limit for heptafluorobutyrates in negative CI-MS was on the order of 1 fg (10−15 g).

In vitro mutagenicity of valepotriates.

Valepotriates are epoxide-bearing triesters of the monoterpene alcohol 4,7-dimethylcyclopenta-(c)-pyrane isolated from the roots of several Valerianacae species. They are regarded as the main tranquilizing constituents of these drugs.Although the valepotriates valtrate/isovaltrate (VAL) and dihydrovaltrate (DH-VAL) showed a strong alkylating activity against the nucleophilic agent 4-(p-nitrobenzyl)-pyridine (NBP), they were not clearly mutagenic for the strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium or for the strains WP2 and WP2 uvrA− of Escherichia coli in the absence of a metabolic activation system (S9-mix). However, the valepotriates were mutagenic for TA100, WP2 and WP2 uvrA− at concentrations up to about 1.0 μmole/plate when S9-mix was added to the test system. With more than 1 μmole/plate the valepotriates were toxic in the presence of a metabolic activation system for all strains tested. The mutagenicity of the valepotriates was inversely related to the protein content of the S9-mix used. The mutagenicity and toxicity of the valepotriates could be inhibited when the S9-mix was preincubated with the esterase inhibitor paraoxon (1 mM) for 5 min before the test compounds and bacteria were added. Therefore, bioactivation of the valepotriates by an enzymatic hydrolysis of their ester groups is considered. This could be proven by activating the valepotriates with purified esterase.

Investigation, with the Salmonella/microsome test, of psychopharmaceuticals used in meat production

Azaperone, acepromazine, xylazine and diazepam were tested for mutagenic activity. To screen these veterinary drugs we used the Salmonella/microsome test on 5 histidine-auxotrophic strains, with and without metabolic activation. Azaperone and xylazine were found to be weakly mutagenic.

GC-MS-Analyse von Polyhydroxyfettsäuren unter Verwendung der Chemischen Ionisation

SummaryPolyhydroxy fatty acids were analysed by GC-MS after trimethylsilylation of the methyl esters. Comparison of various ionization techniques in the mass spectrometric analysis showed that chemical ionization was superior to electron impact ionization. Chemical ionization with methane or isobutane as reactant gas yielded all the necessary ions for structure determination with high relative intensity. The molecular weight can be determined with certainty by the ion group M+H, M−15, M+H−90 using both reactant gases. With methane an intense M+29 is additionally observed. The number of hydroxy groups is obtained from the ionic series M+H−90. The position of the hydroxy groups is given by the fragments after α-cleavage of the fatty acid chain. The polyhydroxy fatty acids can be quantitated in trace amounts in complex mixtures by means of multiple ion detection utilizing the group specific M+H−90 ions.ZusammenfassungDie GC-MS-Analyse von Polyhydroxyfettsäuren wird in Form ihrer trimethylsilylierten Methylester durchgeführt. Der Vergleich verschiedener Ionisierungstechniken in der massenspektrometrischen Bestimmung dieser Substanzgruppe zeigt, daß die Chemische Ionisation der Elektronenstoßionisation überlegen ist. Die Chemische Ionisation mit Methan oder Iso-Butan als Reaktantgas liefert alle für eine Strukturbestimmung wichtigen Ionen mit hoher relativer Intensität. Das Molekulargewicht ist durch die Ionengruppe M+H, M−15, M+H−90 bei Verwendung beider Reaktantgase sicher bestimmbar. Mit Methan wird zusätzlich ein intensives M+29 beobachtet. Die Anzahl der Hydroxygruppen ergibt sich aus der Ionenreihe M+H−n×90. Die Stellung der Hydroxygruppen wird durch die durch α-Spaltung der Kette entstehenden Fragmente erkennbar.Über die gruppenspezifischen M+H−90-Ionen lassen sich die Polyhydroxyfettsäuren mit Hilfe der Mehrfachmassenregistrierung in komplexen Gemischen im Spurenbereich nachweisen.

Enzymatische Oxydation von Linolsäure in Cucurbitaceae

SummaryThe enzymatic oxidation of linoleic acid was studied in raw homogenates of various Cucurbitaceae, such as honeydew-, ogden- and water melons, as well as in pumpkin and zucchini. Analysis of fatty acid composition showed that the main substrates of enzymatic oxidation, namely linoleic and linolenic acid, are present in the cucurbitaceae examined in amounts ranging from 20–60%.Oxygen uptake after the addition of linoleic acid to the homogenates was used as a preliminary measurement of substrate oxidation.The optimum pH for linoleic acid oxidation as determined by this method is from weakly acidic to neutral.After one hour incubation of linoleic acid with fruit homogenates at 25 °C the oxidation products were extracted, and separated and purified by thin layer chromatography. Structure analysis was performed by means of glass capillary gas chromatography mass spectrometry utilizing electron impact and chemical ionization with various reactant gases.The enzymatic oxidation products of linoleic acid in fruit homogenates were identified as mono-, di-, trihydroxy, and oxo compounds of unsaturated octadecanoic acids. The primary oxidation products of linoleic acid oxidation, the hydroperoxides, could not be detected.ZusammenfassungIn der vorliegenden Arbeit wurde die enzymatische Oxydation der Linolsäure durch Fruchthomogenate verschiedenerCucurbitaceae —Honig-, Ogen- und Wassermelone, sowie Kürbis und Zucchini - untersucht. Eine Analyse der Fettsäurezusammensetzung zeigte, daß die wichtigsten Substrate der enzymatischen Fettoxydation, Linol- und Linolensäure, im extrahierbaren Gesamtfett des Fruchtfleisches der hier untersuchtenCucurbitaceae in Mengen von 20–60% enthalten sind.Als Hinweis für eine Substratoxydation diente zunächst die Messung der Sauerstoffaufnahme nach Zugabe von Linolsäure zu den Fruchthomogenaten. Das mit dieser Methode ermittelte pH-Optimum der Linolsäureoxydation liegt im schwach sauren bis neutralen Bereich. Nach Incubation der Fruchtfleischhomogenate mit Linolsäure (1 Std, 25 °C) wurden die Oxydationsprodukte extrahiert, dünnschichtchromatographisch getrennt und gereinigt. Ihre Strukturaufklärung erfolgte mit Hilfe der Glascapillargaschromatographie-Massenspektrometrie mit Elektronenstoßionisation und chemischer Ionisation mit verschiedenen Reaktantgasen.Als Folgeprodukte der enzymatischen Linolsäureoxydation konnten in Fruchthomogenaten von den hier untersuchtenCucurbitaceae Mono-, Di-, Trihydroxy-und Oxo-Verbindungen nachgewiesen werden. Die Primärprodukte der Linolsäureoxydation, die Hydroperoxide, konnten von uns nicht gefunden werden.

Untersuchungen zur Mutagenität von Zuckercouleuren

SummaryIn the present study commercially available caustic and ammoniated caramel colours were tested for their mutagenic potential using the Ames assay. The test was performed using the standard test strainsSalmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without a metabolic activation system (S9-mix). Furthermore, a special preincubation procedure without metabolic activation system was applied. None of the tested caramel colours showed any mutagenic effect in the Ames test.ZusammenfassungIn der vorliegenden Arbeit wurden vier kaustische und drei Ammoniakcouleure auf ihre mutagene Wirkung mit Hilfe des Ames-Tests untersucht. Dazu wurden die StandardteststämmeSalmonella typhimurium TA 1535, TA 1537, TA 98 und TA 100 eingesetzt. Sowohl mit als auch ohne metaboler Aktivierung konnte kein signifikanter Anstieg in der Mutationsrate festgestellt werden. Auch eine Vorinkubation erbrachte für die hier getesteten Zuckercouleure keine Hinweise auf eine mögliche Mutagenität im Ames-Test.In the present study commercially available caustic and ammoniated caramel colours were tested for their mutagenic potential using the Ames assay. The test was performed using the standard test strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without a metabolic activation system (S9-mix). Furthermore, a special preincubation procedure without metabolic activation system was applied. None of the tested caramel colours showed any mutagenic effect in the Ames test.

Untersuchungen zur Mutagenität von Fleischaromen mit natürlichen und/oder naturidentischen Aromastoffen

SummaryThe mutagenic potential of twenty commercially available meat flavours from three different producers was determined using the Ames test. The flavours contained natural and/or nature-identical components as recorded on their label. Twelve flavours showed mutagenic activity with at least one of the four employed test strains (TA1535, TA100, TA97, and TA98). Flavours containing natural components yielded positive results in this mutation test in all cases but one. On the other hand flavours produced from nature-identical substances with one exception did not demonstrate mutagenic effects.ZusammenfassungZwanzig käufliche Fleischaromen wurden mit Hilfe des Ames-Tests mit den StämmenSalmonella typhimurium TA1535, TA100, TA97 und TA98 auf ihre mutagenen Eigenschaften untersucht. Die Aromen enthielten laut Kennzeichnung natürliche und/oder naturidentische Aromastoffe. Ohne Verstoffwechslung erwiesen sich zwölf der untersuchten Fleischaromen als mutagen; eine dosisabhängige Erhöhung der Revertantenzahlen trat in mindestens einem der vier Teststämme auf. Die Zugabe von Rattenleber-S9-Fraktionen führte zu einer Verringerung der Mutationshäufigkeit. Fleischaromen, die positive Ergebnisse im Ames-Test ergaben, enthielten bis auf eines natürliche Aromastoffe. Von den sechs untersuchten Aromen mit nur naturidentischen Aromastoffen zeigte lediglich eines mutagene Effekte.