The influence of the SULT1A status - wild-type, knockout or humanized - on the DNA adduct formation by methyleugenol in extrahepatic tissues of mice.

Methyleugenol, present in herbs and spices, has demonstrated carcinogenic activity in the liver and, to a lesser extent, in extrahepatic tissues of rats and mice. It forms DNA adducts after hydroxylation and sulphation. As previously reported, hepatic DNA adduct formation by methyleugenol in mice is strongly affected by their sulphotransferase (SULT) 1A status. Now, we analysed the adduct formation in extrahepatic tissues. The time course of the adduct levels was determined in transgenic (tg) mice, expressing human SULT1A1/2, after oral administration of methyleugenol (50 mg per kg body mass). Nearly maximal adduct levels were observed 6 h after treatment. They followed the order: liver > caecum > kidney > colon > stomach > small intestine > lung > spleen. We then selected liver, caecum, kidney and stomach for the main study, in which four mouse lines [wild-type (wt), Sult1a1-knockout (ko), tg, and humanized (ko-tg)] were treated with methyleugenol at varying dose levels. In the liver, caecum and kidney, adduct formation was nearly completely dependent on the expression of SULT1A enzymes. In the liver, human SULT1A1/2 led to higher adduct levels than mouse Sult1a1, and the effects of both enzymes were approximately additive. In the caecum, human SULT1A1/2 and mouse Sult1a1 were nearly equally effective, again with additive effects in tg mice. In the kidney, only human SULT1A1/2 played a role: no adducts were detected in wt and ko mice even at the highest dose tested and the adduct levels were similar in tg and ko-tg mice. In the stomach, adduct formation was unaffected by the SULT1A status. IN CONCLUSION: (i) the SULT1A enzymes only affected adduct formation in those tissues in which they are highly expressed (mouse Sult1a1 in the liver and caecum, but not in the kidney and stomach; human SULT1A1/2 in the liver, caecum and kidney, not in the stomach of tg mice and humans), indicating a dominating role of local bioactivation; (ii) the additivity of the effects of both enzymes in the liver and caecum implies that the enzyme level was limiting in the adduct formation; (iii) SULT1A forms dominated the activation of methyleugenol in several tissues, but non-Sult1a1 forms or SULT-independent mechanisms were involved in its adduct formation in the stomach.

Isolation of catechin-converting human intestinal bacteria.

AIMS: To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet. METHODS AND RESULTS: Two bacterial strains, rK3 and aK2, were isolated from an epicatechin-converting human faecal suspension. The isolates catalysed individual steps in the degradation of ⁻-epicatechin and ⁺-catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C-ring of both ⁻-epicatechin and ⁺-catechin giving rise to 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol to 5-(3,4-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific. CONCLUSIONS: The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability. SIGNIFICANCE AND IMPACT OF THE STUDY: The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin-converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.

Bacterial response to eukaryotic cells. Analysis of differentially expressed proteins using nano liquid chromatography-electrospray ionization tandem mass spectrometry.

Host-bacteria interactions have mostly been investigated with regard to the host response or to activities of pathogenic bacteria. In contrast, we aim to identify reactions of non-pathogenic bacteria that result from their contact with host cells of the gastrointestinal tract. In a proteomic approach, the response of non-pathogenic human Escherichia coli bacteria on gut epithelial cells (rat IEC-6) was investigated in an in vitro co-culture model. For this purpose, a sensitive analytical procedure was developed based on the identification of two-dimensional polyacrylamide gel electrophoresis separated proteins by online nanoLC-electrospray ionization MS/MS using a quadrupole time-of-flight tandem mass spectrometer for accurate mass determination. We demonstrate here the efficiency of this technique by the identification of a total of 43 differentially expressed proteins, out of which 25 were up-regulated and 18 were down-regulated. They represent a wide range of molecular weight and different metabolic and physiological functions.

Degradation of quercetin and luteolin by Eubacterium ramulus.

The degradation of the flavonol quercetin and the flavone luteolin by Eubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by (1)H and (13)C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation of E. ramulus. The degradation of luteolin by resting cells of E. ramulus resulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways in E. ramulus start with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.

Benzylic hydroxylation of 1-methylpyrene and 1-ethylpyrene by human and rat cytochromes P450 individually expressed in V79 Chinese hamster cells.

Alkyl-substituted polycyclic aromatic hydrocarbons may be metabolized to highly reactive benzylic sulfuric acid esters via benzylic hydroxylation and subsequent sulfonation. We have studied the benzylic hydroxylation of 1-methylpyrene (MP), a hepatocarcinogen in rodents, and 1-ethylpyrene (EP), whose benzylic hydroxylation would produce a secondary alcohol (alpha-HEP), in contrast to the primary alcohol (alpha-HMP) formed from MP. The hydrocarbons were incubated with hepatic microsomal preparations from humans and rats, as well as with V79-derived cell lines engineered for the expression of individual cytochrome P450 (CYP) forms from human (1A1, 1A2, 1B1, 2A6, 2E1, 3A4) and rat (1A1, 1A2, 2B1). All microsomal systems and CYP-expressing cell lines used, but not CYP-deficient V79 cells, showed biotransformation of both hydrocarbons. Formation of the benzylic alcohol was detected in each case. alpha-HMP and its oxidation product, 1-pyrenylcarboxylic acid (COOH-P), accounted for a major part of the total amount of the metabolites formed from MP in the presence of human liver microsomes (38-64%) and cells expressing human 3A4, 2E1 or 1B1 (80-85%). Likewise, cells expressing human 1A1 showed a higher contribution of alpha-HMP and COOH-P to the total metabolites (45%) than cells expressing the orthologous enzyme of the rat (3%). EP was metabolized at a higher rate and with modified regioselectivity compared with MP, although omega-hydroxylation of the side chain was not detected with the cell lines and only accounted for a small percent of the biotransformation by the microsomal preparations. The highest contributions of alpha-HEP to the total metabolites from EP were detected with the cells expressing human 1A1, 1B1 and 3A4 (38-51%). alpha-HEP accounted for 16% of the metabolites formed in the presence of human hepatic microsomes. Thus, benzylic hydroxylation is a major initial step in the metabolism of MP and EP. This pathway appears to be even more important in humans than in rats. Previously, we had shown that the second step of the activation, the sulfonation of alpha-HMP and alpha-HEP, is also efficiently catalysed by various forms of human sulfotransferases.

Flavonols and flavones of parsley cell suspension culture change the antioxidative capacity of plasma in rats.

The flavonoid aglycones from an illuminated parsley (Petroselinum crispum (Mill.) Nym.) cell suspension culture were identified and quantified as the flavones apigenin, luteolin and chrysoeriol and the flavonols kaempferol, quercetin and isorhamnetin. Flavonoid extracts from these cultures were purified by solid phase extraction from RP C-18 phase and given by gavage to rats. Only extract from illuminated culture increased the antioxidative capacity (AOC) of blood plasma temporarily with maximum values after 1 h. It is concluded that the course of AOC reflects changes in the plasma content of flavonoids.

Chiral inversion of 1-hydroxyethylpyrene enantiomers mediated by enantioselective sulfotransferases.

The benzylic alcohol 1-hydroxyethylpyrene (1-HEP) is activated to a mutagen by sulfotransferases. The sulfuric acid ester formed is difficult to detect, as it is rapidly hydrolysed back to the alcohol. Incubation of the individual enantiomers of 1-HEP with human hydroxysteroid sulfotransferase (hHST) or estrogen sulfotransferase (hEST), expressed in bacteria, led to the formation of the other enantiomer. The rates of sulfation were determined from the initial rates of chiral inversion of the alcohol, knowing that hydrolysis follows an SN1 mechanism and therefore produces racemic alcohol. hEST showed high enantioselectivity for S-1-HEP, whereas hHST strongly preferred the R-enantiomer. The rates of sulfation of the preferred enantiomers were high, similar to those for the prototype substrates of hEST (beta-estradiol) and hHST (dehydroepiandrosterone). Moreover, after a 30-min incubation of S-1-HEP with hEST, 95% of the recovered alcohol showed the R-configuration, indicating that several cycles of sulfation and hydrolysis had led to the depletion of one enantiomer and to the enrichment of the other enantiomer.

[Mass spectrometric structure analysis of fatty acid mixtures from biological material after gas capillary chromatographical separation]. / Massenspektrometrische Strukturanalyse von Fettsäuregemischen aus biologischem Material nach kapillargaschromatographischer Trennung.

The identification of mixtures of fatty acids from biological materials is possible by electron impact ionization mass spectra of methyl esters after their capillary gas chromatographic separation. Mass spectra of pyrrolidine derivatives are used for the determination of double bond positions in unsaturated fatty acids. 97 different fatty acids (saturated, unsaturated, branched, cyclic, hydroxy, oxo, epoxy and methoxy) and other compounds (alkanes, halogens, phthalates, ketones, aldehydes) were identified in yeast and bacterial biomasses, lipid-containing animal tissues and human sera as well as fish and plant oils (77 preparations).