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Juices of Brassica vegetables are mutagenic and form characteristic DNA adducts in bacteria and mammalian cells. In this study, we examined whether such adducts are also formed in vivo in animal models. Rats fed raw broccoli ad libitum in addition to normal laboratory chow for 5 weeks showed one major adduct spot and sometimes an additional minor adduct spot in liver, kidney, lung, blood and the gastrointestinal tract, as determined by 32P-postlabelling/thin-layer chromatography. Adducts with the same chromatographic properties were formed when herring sperm DNA (or dG-3’-phosphate) was incubated with 1-methoxy-3-indolylmethyl glucosinolate (phytochemical present in Brassica plants), in the presence of myrosinase (plant enzyme that hydrolyses glucosinolates to bioactive breakdown products). UPLC–MS/MS analysis corroborated this finding: 1-Methoxy-3-indolylmethyl-substituted purine nucleosides were detected in the hepatic DNA of broccoli-fed animals, but not in control animals. Feeding raw cauliflower led to the formation of the same adducts. When steamed rather than raw broccoli was used, the adduct levels were essentially unchanged in liver and jejunum, but elevated in large intestine. Due to inactivation of myrosinase by the steaming, higher levels of the glucosinolates may have reached the large bowl to be activated by glucosidases from intestinal bacteria. In conclusion, the consumption of common Brassica vegetables can lead to the formation of substantial levels of DNA adducts in animal models. The adducts can be attributed to a specific phytochemical, neoglucobrassicin (1-methoxy-3-indolylmethyl glucosinolate).

The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (+)-taxifolin but not that of (−)-taxifolin. The K m for (+)-taxifolin was 6.4 ± 0.8 μM, and the V max was 108 ± 4 μmol min −1 (mg protein) −1 . The enzyme also isomerized (+)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus . Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439–1442, 2014, http://dx.doi.org/10.1002/anie.201306952 ), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus . IMPORTANCE Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus . Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria.

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.

Gut bacteria play a crucial role in the metabolism of dietary flavonoids and thereby influence the bioactivity of these compounds in the host. The intestinal Lachnospiraceae strain CG19-1 and Eubacterium cellulosolvens are able to deglycosylate C- and O-coupled flavonoid glucosides. Growth of strain CG19-1 in the presence of the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) led to the induction of two proteins (DfgC, DfgD). Heterologous expression of the encoding genes (dfgC, dfgD) in Escherichia coli revealed no C-deglycosylating activity in the resulting cell extracts but cleavage of flavonoid O-glucosides such as daidzin (daidzein 7-O-glucoside). The recombinant DfgC and DfgD proteins were purified and characterized with respect to their quaternary structure, substrate and cofactor specificity. The products of the corresponding genes (dfgC, dfgD) from E. cellulosolvens also catalysed the O-deglycosylation of daidzin following their expression in E. coli. In combination with three recombinant proteins encoded by adjacent genes in E. cellulosolvens (dfgA, dfgB, dfgE), DfgC and DfgD from E. cellulosolvens catalysed the deglycosylation of the flavone C-glucosides homoorientin (luteolin 6-C-glucoside) and isovitexin (apigenin 6-C-glucoside). Even intact cells of E. coli expressing the five E. cellulosolvens genes cleaved these flavone C-glucosides and, also, flavonoid O-glucosides to the corresponding aglycones.

Gut bacteria play a crucial role in the metabolism of dietary flavonoids and thereby influence the bioactivity of these compounds in the host. The intestinal Lachnospiraceae strain CG19-1 and Eubacterium cellulosolvens are able to deglycosylate C- and O-coupled flavonoid glucosides. Growth of strain CG19-1 in the presence of the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) led to the induction of two proteins (DfgC, DfgD). Heterologous expression of the encoding genes (dfgC, dfgD) in Escherichia coli revealed no C-deglycosylating activity in the resulting cell extracts but cleavage of flavonoid O-glucosides such as daidzin (daidzein 7-O-glucoside). The recombinant DfgC and DfgD proteins were purified and characterized with respect to their quaternary structure, substrate and cofactor specificity. The products of the corresponding genes (dfgC, dfgD) from E. cellulosolvens also catalysed the O-deglycosylation of daidzin following their expression in E. coli. In combination with three recombinant proteins encoded by adjacent genes in E. cellulosolvens (dfgA, dfgB, dfgE), DfgC and DfgD from E. cellulosolvens catalysed the deglycosylation of the flavone C-glucosides homoorientin (luteolin 6-C-glucoside) and isovitexin (apigenin 6-C-glucoside). Even intact cells of E. coli expressing the five E. cellulosolvens genes cleaved these flavone C-glucosides and, also, flavonoid O-glucosides to the corresponding aglycones.