Heiko Schneider

Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract.

Abstract From human feces two phenotypically different types of bacteria were isolated on quercetin-3-glucoside as carbon and energy source. Isolates of one type were identified as strains of Enterococcus casseliflavus. They utilized the sugar moiety of the glycoside, but did not degrade the aglycon further. The sugar moiety (4 mM) was fermented to 5.5 ± 2.1 mM formate, 2.1 ± 0.7 mM acetate, 1.6 ± 0.3 mM l-lactate, and 1.3 ± 0.4 mM ethanol. The second type of isolate was identified as Eubacterium ramulus. This organism was capable of degrading the aromatic ring system. Growing cultures of Eubacterium ramulus converted 5 mM quercetin-3-glucoside to 1.7 ± 0.6 mM 3,4-dihydroxyphenylacetic acid, 7.6 ± 1.0 mM acetate, and 4.0 ± 0.4 mM butyrate. Molecular hydrogen, 3,4-dihydroxybenzaldehyde, and ethanol were detected in small amounts. Phloroglucinol was a transient intermediate in the breakdown of quercetin-3-glucoside. Eubacterium ramulus did not grow on the aglycon quercetin or the ring-fission intermediate phloroglucinol, but cleaved the flavonoid ring system when glucose was present as a cosubstrate. The most probable number of quercetin-3-glucoside-degrading bacteria determined in nine human fecal samples was 107–109/g dry mass. Isolates from these experiments were all identified as Eubacterium ramulus.

Anaerobic degradation of flavonoids by Eubacterium ramulus.

Eubacterium ramulus, a quercetin-3-glucoside-degrading anaerobic microorganism that occurs at numbers of approximately 108/g dry feces in humans, was tested for its ability to transform other flavonoids. The organism degraded luteolin-7-glucoside, rutin, quercetin, kaempferol, luteolin, eriodictyol, naringenin, taxifolin, and phloretin to phenolic acids. It hydrolyzed kaempferol-3-sorphoroside-7-glucoside to kaempferol-3-sorphoroside and transformed 3,4-dihydroxyphenylacetic acid, a product of anaerobic quercetin degradation, very slowly to non-aromatic fermentation products. Luteolin-5-glucoside, diosmetin-7-rutinoside, naringenin-7-neohesperidoside, (+)-catechin, and (–)-epicatechin were not degraded. Cell extracts of E. ramulus contained α- and β-d-glucosidase activities, but were devoid of α-l-rhamnosidase activity. Based on the degradation patterns of these substrates, a pathway for the degradation of flavonoids by E. ramulus is proposed.

Degradation of quercetin‐3‐glucoside in gnotobiotic rats associated with human intestinal bacteria

Aim: The two bacterial species, Eubacterium ramulus and Enterococcus casseliflavus, which had previously been isolated from human faeces using the flavonoid quercetin‐3‐glucoside as the growth substrate, were tested for their ability to utilize this compound in vivo.

Sulpho-conjugation of ethanol in humans in vivo and by individual sulphotransferase forms in vitro.

We studied whether ethanol is sulphonated in humans with the perspective of using the urinary excretion of ethyl sulphate after ethanol consumption as a biomarker for SULT (sulphotransferase) activity. We developed a sensitive and selective HPLC-MS/MS method for determining ethyl sulphate in urine. Ten volunteers received a low dose of ethanol (0.1 g/kg of body mass). In general, excretion of ethyl sulphate was maximal in the first or second hour after dosage. Within 8 h, 2.5-6.8 micromol of ethyl sulphate was excreted. A 5-fold increase in the dose of ethanol led to an increase in the amount of ethyl sulphate excreted within 8 h (28-95 micromol) and the presence of this metabolite in urine for at least 24 h. Since ethyl sulphate was still being excreted for a substantial period after the elimination of ethanol, it might be used as a medium-time biomarker for preceding ethanol consumption. We have expressed previously all human SULT forms identified in Salmonella typhimurium. Ethanol sulphonation was studied in cytosolic preparations of these strains. The highest activities were observed with SULT1A2, 1B1 and 1C2, followed by 1A3. Activities were markedly lower with SULT1E1, 1A1 and 2A1, and were negligible with SULT1C1, 2B1a, 2B1b and 4A1. If the expression levels in tissues are additionally taken into account, SULT1A3 might be the predominant form for the sulphonation of ethanol in vivo, although a robust estimate requires further studies. With this limitation, urinary ethyl sulphate excretion appears very promising as a biomarker for SULT activity in vivo.

Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry

Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, and N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction by xenobiotics. Ratios of different caffeine metabolites in urine or other body fluids are frequently used to characterise the individual/actual activity of these enzymes. The common analytical method involves extensive sample preparation, followed by HPLC-UV. The presence of numerous other UV-absorbing chemicals in body fluids affects the sensitivity and selectivity of this method. We have developed an HPLC-electrospray-MS-MS method for the determination of 11 caffeine metabolites and two internal standards after a simple, extractionless preparation. Blank urine, obtained after 5 days on a methylxanthine-free diet, contained small amounts of some caffeine metabolites, but no other components producing any confounding signals. Eleven metabolites and internal standards were recovered at 90 to 110% after addition to the blank urine (0.1 to 2.5 micro M in the final sample involving a 20-fold dilution of urine) in the 0.1-2.5 micro M concentration range. Other metabolites, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU), were detected with similar recovery and precision, but required higher concentrations (3 to 30 micro M). AFMU was completely converted into AAMU by a short alkalisation of urine. The method was explored in six healthy individuals after consuming coffee (4 mg caffeine per kg body mass). These experiments demonstrated the simplicity, high sensitivity and selectivity of the method under conditions used for phenotyping.

Hydroxymethyl-substituted furans: mutagenicity in Salmonella typhimurium strains engineered for expression of various human and rodent sulphotransferases.

5-Hydroxymethylfurfural (HMF) and furfuryl alcohol (FFA) are present in numerous foodstuffs at high levels. FFA is also used for the production of polymers. Both compounds had demonstrated some evidence of carcinogenic activity in 2-year bioassays. We tested these compounds and four congeners for mutagenicity in Salmonella typhimurium TA100 and TA100-derived strains expressing human or rodent sulphotransferases (SULTs). 5-Hydroxymethylfuroic acid, a metabolite of HMF, was not mutagenic in any strain. 3-Hydroxymethylfuran was weakly mutagenic in all strains independently of SULT expression. HMF, 2,5-(bishydroxymethyl)furan (metabolite of HMF), FFA and 5-methyl-FFA were inactive in TA100 but strongly mutagenic when human SULT1C2 was expressed. This form has been detected in ovary, kidney and foetal tissues. Human SULT1A1, SULT1A2 and SULT1A3 as well as murine Sult1a1 and Sult1d1 also activated some hydroxymethyl-substituted furans to varying degrees. Whereas chemically synthesised 5-sulphooxymethylfurfural was mutagenic in TA100, furfuryl sulphate was bacteriotoxic, only leading to marginal increases in the number of revertants. Furfuryl acetate, an uncharged ester of FFA, used as fragrance and food flavouring, was clearly mutagenic. We determined half-life times of 120 min, 20 s and 10 h, respectively, for 5-sulphooxymethylfurfural, furfuryl sulphate and furfuryl acetate at 37°C in water. It is likely that the short lifespan of furfuryl sulphate, together with its charge, led to insufficient penetration of the bacteria when added externally, although it was mutagenic when generated by appropriate SULTs from FFA within the cell.

Regulatory toxicology in the twenty-first century: challenges, perspectives and possible solutions

Abstract The advent of new testing systems and “omics”-technologies has left regulatory toxicology facing one of the biggest challenges for decades. That is the question whether and how these methods can be used for regulatory purposes. The new methods undoubtedly enable regulators to address important open questions of toxicology such as species-specific toxicity, mixture toxicity, low-dose effects, endocrine effects or nanotoxicology, while promising faster and more efficient toxicity testing with the use of less animals. Consequently, the respective assays, methods and testing strategies are subject of several research programs worldwide. On the other hand, the practical application of such tests for regulatory purposes is a matter of ongoing debate. This document summarizes key aspects of this debate in the light of the European “regulatory status quo”, while elucidating new perspectives for regulatory toxicity testing.

Die Vorgehensweise des Bundesinstitutes für Risikobewertung bei der Abschätzung der dermalen Absorption von Wirkstoffen in Pflanzenschutzmitteln und Biozidprodukten

ZusammenfassungFür die Bewertung des gesundheitlichen Risikos von Pflanzenschutzmitteln (PSM) und Biozidprodukten (BP) ist die korrekte Abschätzung der Aufnahme der darin enthaltenen Wirkstoffe über die Haut erforderlich. In dieser Veröffentlichung wird dargestellt, wie das Bundesinstitut für Risikobewertung (BfR), in Abhängigkeit von den verfügbaren Daten, die dermale Absorption von Wirkstoffen in PSM und BP ableitet. Dabei finden zwei neue Bewertungsleitfäden der EFSA und der OECD Verwendung. Grundlegende Prinzipien der Studienauswertung werden ebenso beschrieben wie Möglichkeiten zur Abschätzung der Hautabsorption, wenn keine produktspezifischen experimentellen Daten vorliegen. Die Publikation dieser Vorgehensweise stellt einen ersten Schritt für die Harmonisierung der Bewertung in den Zulassungsverfahren von PSM und BP dar.AbstractA correct estimate of dermal absorption of the contained active ingredients is needed for the health risk assessment of plant protection and biocidal products. In this paper, the approach is reported that is currently taken by the Federal Institute for Risk Assessment to derive the dermal absorption rate of active substances in plant protection or biocidal products, depending on the available data. It is explained what use is made of two new guidance documents that were recently released by EFSA and OECD. Basic principles of the assessment of dermal absorption studies are described but also opportunities for estimations in the absence of product-specific experimental data. This publication is considered a first step towards harmonisation of assessment in the authorisation processes for plant protection and biocidal products.