Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products

The metabolism of chlorogenic acid, naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-(3-hydroxyphenyl)-propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols.     

Studies on flavonoid metabolism. Metabolism of (+)-catechin in the guinea pig

1. Administration of (+)-catechin to the guinea pig gives rise to a number of phenolic acids and lactones, which have been identified by chromatographic and spectrophotometric methods. The major phenolic acid metabolite is m-hydroxybenzoic acid and the major lactone metabolite is delta-(3-hydroxyphenyl)-gamma-valerolactone. 2. The phenolic acid and lactone metabolites are excreted in both free and conjugated forms, including their glucuronides and to a lesser degree their ethereal sulphates. 3. Administration of certain of the metabolites isolated has permitted certain sequential relationships of these intermediates to be established. 4. Degradation of (+)-catechin in the guinea pig is effected at least in part by the gut microflora and is suppressed by aureomycin plus phthaloyl-sulphathiazole.     

The fate of olive oil polyphenols in the gastrointestinal tract: Implications of gastric and colonic microflora-dependent biotransformation

We have conducted a detailed investigation into the absorption, metabolism and microflora-dependent transformation of hydroxytyrosol (HT), tyrosol (TYR) and their conjugated forms, such as oleuropein (OL). Conjugated forms underwent rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of free HT and TYR entering the small intestine. Both HT and TYR transferred across human Caco-2 cell monolayers and rat segments of jejunum and ileum and were subject to classic phase I/II biotransformation. The major metabolites identified were an O-methylated derivative of HT, glucuronides of HT and TYR and a novel glutathionylated conjugate of HT. In contrast, there was no absorption of OL in either model. However, OL was rapidly degraded by the colonic microflora resulting in the formation of HT. Our study provides additional information regarding the breakdown of complex olive oil polyphenols in the GI tract, in particular the stomach and the large intestine.     

The fate of olive oil polyphenols in the gastrointestinal tract: implications of gastric and colonic microflora-dependent biotransformation

We have conducted a detailed investigation into the absorption, metabolism and microflora-dependent transformation of hydroxytyrosol (HT), tyrosol (TYR) and their conjugated forms, such as oleuropein (OL). Conjugated forms underwent rapid hydrolysis under gastric conditions, resulting in significant increases in the amount of free HT and TYR entering the small intestine. Both HT and TYR transferred across human Caco-2 cell monolayers and rat segments of jejunum and ileum and were subject to classic phase I/II biotransformation. The major metabolites identified were an O-methylated derivative of HT, glucuronides of HT and TYR and a novel glutathionylated conjugate of HT. In contrast, there was no absorption of OL in either model. However, OL was rapidly degraded by the colonic microflora resulting in the formation of HT. Our study provides additional information regarding the breakdown of complex olive oil polyphenols in the GI tract, in particular the stomach and the large intestine.     

Deglycosylation of puerarin and other aromatic C-glucosides by a newly isolated human intestinal bacterium

The human intestinal microbiota may influence the fate of bioactive polyphenols, such as the isoflavone puerarin (daidzein 8-C-glucoside), following their oral intake. Faecal suspensions from 19 healthy subjects were tested for their ability to C-deglycosylate puerarin. Only one of these catalysed this reaction. A rod-shaped Gram-positive bacterium, strain CG19-1, capable of deglycosylating puerarin to daidzein was isolated from the corresponding suspension. However, the strictly anaerobic isolate was unable to utilize puerarin as sole carbon and energy source nor any of the tested carbohydrates. Comparative 16S rRNA gene sequence analysis indicated that strain CG19-1 is a new species of the Lachnospiraceae. Strain CG19-1 also converted other aromatic C-glucosides in addition to puerarin. The xanthone C-glucoside mangiferin was deglycosylated to norathyriol. The flavone C-glucosides homoorientin and vitexin were degraded to 3-(3,4-dihydroxyphenyl)propionic acid via luteolin and 3-(4-hydroxyphenyl)propionic acid respectively. In addition, strain CG19-1 converted flavonoid O-glucosides, but at rates that were lower than those of the C-glucosides tested. The isolate deglycosylated the isoflavone O-glucosides daidzin and genistin to daidzein and genistein respectively. Several O-glucosides of the flavones luteolin and apigenin undergoing deglycosylation were subsequently cleaved to 3-(3,4-dihydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)propionic acid respectively. Moreover, strain CG19-1 cleaved both O-desmethylangolensin and 6'-hydroxy-O-desmethylangolensin to yield 2-(4-dihydroxyphenyl)propionic acid. The corresponding cleavage product, resorcinol, was only observed for O-desmethylangolensin.     

Roles of glucuronidation and UDP-glucuronosyltransferases in xenobiotic bioactivation reactions

Glucuronide conjugates represent one of the major types of naturally occurring phase 2 metabolites of xenobiotics and endobiotics. The process underlying their formation, glucuronidation, is normally considered detoxifying, because glucuronides usually possess less intrinsic biological or chemical activity than their parent aglycones and they are rapid excreted. However, a number of glucuronide conjugates are known that are active and may contribute to pharmacological activities or toxicities associated with their parent compounds. These include two classes of glucuronides with electrophilic chemical reactivity (N-O-glucuronides of hydroxamic acids and acyl glucuronides of carboxylic acids) and several types of glucuronides that impart biological effects through non-covalent interactions (morphine 6-O-glucuronide, retinoid glucuronides, and D-ring glucuronides of estrogens). Glucuronides may thus contribute to clinically significant effects, including environmental arylamine-induced carcinogenesis, drug hypersensitivity and other toxicities associated with carboxylic acid drugs, morphine analgesia, and cholestasis from estrogens. This review summarizes the rat and human UDP-glucuronosyltransferases that may be involved in the formation of bioactive glucuronides, including their substrate- and tissue-specificity and genetic and environmental influences on their activity. This knowledge may be useful for enhancing the therapeutic efficacy and minimizing the risk of adverse effects associated with xenobiotics that undergo bioactivating glucuronidation reactions.     

The metabolism of dietary polyphenols and the relevance to circulating levels of conjugated metabolites

Berry extracts rich in anthocyanins have been linked to protective effects including the modulation of age-related neurological dysfunction and the improvement of the resistance of red blood cells against oxidative stress in vitro . In this study the bioavailability, metabolism and elimination of polyphenols from blackcurrant juice, rich in anthocyanins, flavonols, and hydroxycinnamates, were investigated. The four major native anthocyanidin glycosides of blackcurrant juice, delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside, were detected and identified in low amounts by HPLC and LC-MS in plasma and urine post-ingestion. Elimination of the anthocyanins was fast (maximum excretion after 1 h) and plasma levels (0-128.6 nmol/l) and total urinary excretion (0.07-1.35 mg; 0.007-0.133% of the dose ingested) were low. Most significantly, of the hydroxycinnamates, conjugated and free ferulic, isoferulic, p -coumaric, sinapic and vanillic acids were identified in plasma and urine, using GC-MS techniques. Quercetin and kaempferol (as glucuronides) and the proposed colonic metabolite of quercetin, 3-hydroxyphenylacetic acid, were detectable in a minority of subjects. Increased daily urinary hippuric, 4-hydroxyhippuric and 3-hydroxyhippuric acid levels were also observed post-ingestion in all volunteers.     

The Metabolism of Dietary Polyphenols and the Relevance to Circulating Levels of Conjugated Metabolites

Berry extracts rich in anthocyanins have been linked to protective effects including the modulation of age-related neurological dysfunction and the improvement of the resistance of red blood cells against oxidative stress in vitro . In this study the bioavailability, metabolism and elimination of polyphenols from blackcurrant juice, rich in anthocyanins, flavonols, and hydroxycinnamates, were investigated. The four major native anthocyanidin glycosides of blackcurrant juice, delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside, were detected and identified in low amounts by HPLC and LC-MS in plasma and urine post-ingestion. Elimination of the anthocyanins was fast (maximum excretion after 1 h) and plasma levels (0-128.6 nmol/l) and total urinary excretion (0.07-1.35 mg; 0.007-0.133% of the dose ingested) were low. Most significantly, of the hydroxycinnamates, conjugated and free ferulic, isoferulic, p -coumaric, sinapic and vanillic acids were identified in plasma and urine, using GC-MS techniques. Quercetin and kaempferol (as glucuronides) and the proposed colonic metabolite of quercetin, 3-hydroxyphenylacetic acid, were detectable in a minority of subjects. Increased daily urinary hippuric, 4-hydroxyhippuric and 3-hydroxyhippuric acid levels were also observed post-ingestion in all volunteers.     

Lactobacillus plantarum IFPL935 impacts colonic metabolism in a simulator of the human gut microbiota during feeding with red wine polyphenols

The colonic microbiota plays an important role in the bioavailibility of dietary polyphenols. This work has evaluated the impact on the gut microbiota of long-term feeding with both a red wine polyphenolic extract and the flavan-3-ol metabolizer strain Lactobacillus plantarum IFPL935. The study was conducted in the dynamic Simulator of the Human Intestinal Microbial Ecosystem (SHIME). The feeding of the gut microbiota model with red wine polyphenols caused an initial decrease in the counts of total bacteria in the ascending colon (AC), with Bacteroides, Clostridium coccoides/Eubacterium rectale and Bifidobacterium being the most affected bacterial groups. The bacterial counts recovered to initial numbers faster than the overall microbial fermentation and proteolysis, which seemed to be longer affected by polyphenols. Addition of L. plantarum IFPL935 helped to promptly recover total counts, Lactobacillus and Enterobacteriaceae and led to an increase in lactic acid formation in the AC vessel at the start of the polyphenol treatment as well as butyric acid in the transverse (TC) and descending (DC) vessels after 5 days. Moreover, L. plantarum IFPL935 favoured the conversion in the DC vessel of monomeric flavan-3-ols and their intermediate metabolites into phenylpropionic acids and in particular 3-(3′-hydroxyphenyl)propionic acid. The results open the possibilities of using L. plantarum IFPL935 as a food ingredient for helping individuals showing a low polyphenol-fermenting metabotype to increase their colonic microbial capacities of metabolizing dietary polyphenols.     

Nutraceuticals: facts and fiction

Epidemiological studies show a link between the consumption of plant-derived foods and a range of health benefits. These benefits have been associated, at least partially, to some of the phytochemical constituents, and, in particular, to polyphenols. In the last few years, nutraceuticals have appeared in the market. These are pharmaceutical forms (pills, powders, capsules, vials, etc.) containing food bioactive compounds as active principles. The bioactive phytochemicals have become a very significant source for nutraceutical ingredients. Scientific research supports the biological activity of many of these food phytochemicals, but the health claims attributed to the final marketed nutraceutical products have often little or doubtful scientific foundation. This is due to the fact that a lot of the scientific evidence is derived from animal testing and in vitro assays, whereas human clinical trials are scarce and inconclusive. Some key issues such as bioavailability, metabolism, dose/response and toxicity of these food bioactive compounds or the nutraceuticals themselves have not been well established yet. Amongst the phytochemicals, several groups of polyphenols (anthocyanins, proanthocyanidins, flavanones, isoflavones, resveratrol and ellagic acid) are currently used in the nutraceutical industry. In this report, we have reviewed the most recent scientific knowledge on the bioavailability and biological activity of these polyphenols ('fact'), as well as the health claims (which are not always supported by scientific studies) ascribed to the polyphenols-containing nutraceuticals ('fiction'). The in vitro antioxidant capacity, often used as a claim, can be irrelevant in terms of in vivo antioxidant effects. Bioavailability, metabolism, and tissue distribution of these polyphenols in humans are key factors that need to be clearly established in association to the biological effects of these polyphenols-containing nutraceuticals. The future trends of phytochemistry research regarding nutraceuticals are discussed.     

Digestive end products release pancreatic enzymes from particulate cellular pools, particularly zymogen granules

The effects of various amino acids and phosphorylated forms of glucose on the release of digestive enzymes from particulate cellular pools, particularly zymogen granules, were evaluated in rat pancreas. Whole tissue homogenates, as well as zymogen granules isolated either by differential centrifugation in 0.3 M sucrose or by preparation in buffered sucrose and subsequent centrifugation in a Percoll gradient, were studied. The basic amino acids L-arginine and L-lysine, sites of tryptic cleavage, caused the release of trypsinogen, but not chymotrypsinogen, whereas the aromatic amino acids L-phenylalanine and L-tryptophan, sites of chymotryptic cleavage, caused release of both trypsinogen and chymotrypsinogen. Neither led to the release of the starch-splitting enzyme amylase. All effects occurred within the range of normal plasma concentrations for these amino acids in the rat. Two amino acids, L-threonine and hydroxy-L-proline, that are not sites of cleavage by trypsin or chymotrypsin, and a nonmammalian amino acid, aminoadipic acid, did not lead to release of trypsinogen, chymotrypsinogen, or amylase. Two phosphorylated forms of glucose, glucose 1-phosphate and glucose 1,6-diphosphate, caused the release of amylase, but of neither trypsinogen nor chymotrypsinogen. Contrary to previous results, D-glucose was without effect, as was glucose 6-phosphate. We propose that certain digestive end products, by direct action on zymogen granules, cause the selective release of the enzymes involved in their evolution from polymeric substrates during digestion.     

Stereochemical aspects of the metabolism of the isomeric methylcyclohexanols and methylcyclohexanones

1. The seven isomeric optically inactive forms of methylcyclohexanol (i.e. 1-, and cis- and trans-2-, -3- and -4-) are excreted by rabbits mainly as glucuronides of the thermodynamically more stable forms of the alcohols. The eighth isomer, cyclohexylmethanol, however, undergoes aromatization in vivo, giving rise to benzoic acid and hippuric acid. The (±)-2-, (±-3- and 4-methylcyclohexanones are reduced in the rabbit and excreted mainly as the glucuronides of the thermodynamically more stable forms of the corresponding methylcyclohexanols. 2. Racemic cis- and trans-2-methylcyclohexanol and 2-methylcyclohexanone are all excreted as conjugated trans-2-methylcyclohexanol. However, when the (±)-cis-alcohol or the (±)-ketone is fed, (+)-trans-2-methylcyclohexanol is excreted, whereas when the (±)-trans-alcohol is fed it is excreted as the (±)-trans-alcohol. 3. Racemic cis- and trans-3-methylcyclohexanol and 3-methylcyclohexanone are all excreted as conjugated racemic cis-3-methylcyclohexanol. cis- and trans-4-Methylcyclohexanol and 4-methylcyclohexanone are all excreted as conjugated trans-4-methylcyclohexanol. 4. The metabolic differences between the various methylcyclohexanols are explicable in terms of their conformations and of Vennesland's (1958) hypothesis of the role of NADH in dehydrogenation reactions.     

6 alpha-glucuronidation of hyodeoxycholic acid by human liver, kidney and small bowel microsomes

Kinetic constants for the glucuronidation of hyodeoxycholic acid in man were determined using microsomal preparations of liver, kidney and small bowel. The affinity of hyodeoxycholic acid for the microsomal hepatic and extrahepatic enzymes was in the same range as previously observed for the monohydroxy bile acid lithocholic acid and about 3-14-times the affinity for the dihydroxy bile acids chenodeoxycholic, deoxycholic and ursodeoxycholic acids. The Vmax values for glucuronidation of hyodeoxycholic acid with hepatic microsomes were 10-30-times higher and with kidney microsomes 50-110-times higher than for the bile acids lacking a 6 alpha-hydroxy group. The site of glucuronidation was determined by gas chromatographic-mass spectrometric analysis of derivatives of products formed after periodate and chromic acid oxidation. Hyodeoxycholic acid glucuronides synthesized with microsomal preparations from the three organs were all found to be conjugated at the 6 alpha position. This has previously been shown to be the site of glucuronidation of endogenous hyodeoxycholic acid glucuronide excreted in urine.     

The Differential Tissue Distribution of the Citrus Flavanone Naringenin Following Gastric Instillation

Citrus flavonoids have been investigated for their biological activity, with both anti-inflammatory and -carcinogenic effects being reported. However, little information is known on the bioavailability of these compounds in vivo. The objectives of this study were to determine the tissue distribution of naringenin after gastric gavage of [³H]-naringenin to rats. Unlabelled naringenin was also used to quantify the levels of naringenin and its major metabolites in tissues and eliminated in the urine and faeces. Significant radioactivity was detected in the plasma as well as all tissues examined 2?h post-gavage. After 18?h, higher levels of radioactivity were retained in plasma and tissues (55% of the administered radioactivity). Investigation of the nature of metabolites, using unlabelled naringenin, revealed that the glucuronides were the major components in plasma, tissues and urine, in addition to the colonic metabolite 3-(4-hydroxyphenyl) propionic acid, detected in the urine. The aglycone was the form extensively retained in tissues after 18?h post-gavage. Total identified metabolites detected after 18?h in most tissues were only 1–5% of the levels detected after 2?h. However, the brain, lungs and heart retained 27, 20 and 11%, respectively, relative to the total metabolites detected at 2?h. While radioactive detection suggests increased levels of breakdown products of naringenin after 18?h versus 2?h, the products identified using unlabelled naringenin are not consistent with this, suggesting that a predominant proportion of the naringenin breakdown products at 18?h are retained as smaller decomposition molecules which cannot yet be identified.     

Biological activities of Apo-canthaxanthinoic acids related to gap junctional communication

Apo-carotenoids with different numbers of conjugated double bonds are formed upon eccentric cleavage of carotenoids. These compounds may exhibit biological activities similar to those of the parent carotenoids or their central cleavage products, the retinoids. 11-Apo-canthaxanthin-11-oic acid, 13-apo-canthaxanthin-13-oic acid, and 14'-apo-canthaxanthin-14'-oic acid, carrying 2, 3, or 5 conjugated double bonds in the polyene chain, respectively, were tested for their effects on gap junctional communication (GJC), on stabilization of connexin43 mRNA, and on the activation of the retinoic acid-beta2 receptor (RAR-beta2 receptor); the effects were compared to those of retinoic acid and 4-oxo-retinoic acid, known to stimulate GJC and to activate the RAR-beta2 receptor. The effects of 4-oxo-retinoic acid were comparable to those of retinoic acid. 4-Oxo-retinoic acid, like retinoic acid, influences the stability of connexin 43 mRNA via elements located in the 3'-UTR. No effects were observed with the short-chain apo-canthaxanthinoic acids. A small but statistically significant induction of GJC and transactivation activity towards the RARbeta2 was found with 14'-apo-canthaxanthin-14'-oic acid. This might be due to biological effects of the compound itself or to biologically active breakdown products. The data suggest that the major biological effects of canthaxanthin on retinoid signaling pathways are related to activities mediated by the products of the central cleavage.     

The differential tissue distribution of the citrus flavanone naringenin following gastric instillation

Citrus flavonoids have been investigated for their biological activity, with both anti-inflammatory and -carcinogenic effects being reported. However, little information is known on the bioavailability of these compounds in vivo. The objectives of this study were to determine the tissue distribution of naringenin after gastric gavage of [³H]-naringenin to rats. Unlabelled naringenin was also used to quantify the levels of naringenin and its major metabolites in tissues and eliminated in the urine and faeces. Significant radioactivity was detected in the plasma as well as all tissues examined 2 h post-gavage. After 18 h, higher levels of radioactivity were retained in plasma and tissues (55% of the administered radioactivity). Investigation of the nature of metabolites, using unlabelled naringenin, revealed that the glucuronides were the major components in plasma, tissues and urine, in addition to the colonic metabolite 3-(4-hydroxyphenyl) propionic acid, detected in the urine. The aglycone was the form extensively retained in tissues after 18 h post-gavage. Total identified metabolites detected after 18 h in most tissues were only 1-5% of the levels detected after 2 h. However, the brain, lungs and heart retained 27, 20 and 11%, respectively, relative to the total metabolites detected at 2 h. While radioactive detection suggests increased levels of breakdown products of naringenin after 18 h versus 2 h, the products identified using unlabelled naringenin are not consistent with this, suggesting that a predominant proportion of the naringenin breakdown products at 18 h are retained as smaller decomposition molecules which cannot yet be identified.     

Cathelicidin family of antimicrobial peptides: proteolytic processing and protease resistance

Cathelicidins are a gene family of antimicrobial peptides produced as inactive precursors. Signal peptidase removes the N-terminal signal sequence, while peptidylglycine alpha-amidating monooxygenase often amidates and cleaves the C-terminal region. Removal of the cathelin domain liberates the active antimicrobial peptide. For mammalian sequences, this cleavage usually occurs through the action of elastase, but other tissue-specific processing enzymes may also operate. Once released, these bioactive peptides are susceptible to proteolytic degradation. We propose that some mature cathelicidins are naturally resistant to proteases due to their unusual primary structures. Among mammalian cathelicidins, proline-rich sequences should resist attack by serine proteases because proline prevents cleavage of the scissile bond. In hagfish cathelicidins, the unusual amino acid bromotryptophan may make the active peptides less susceptible to proteolysis for steric reasons. Such protease resistance could extend the pharmacokinetic lifetimes of cathelicidins in vivo, sustaining antimicrobial activity.     

Characterization of the specificity of arginine-specific gingipains from Porphyromonas gingivalis reveals active site differences between different forms of the enzymes

Porphyromonas gingivalis is a pathogen associated with periodontal disease, and arginine-specific proteases (gingipains-R) from the bacterium are important virulence factors. The specificity of two forms of gingipain-R, HRgpA and RgpB, for substrate positions C-terminal to the cleavage site was analyzed, and notable differences were observed between the enzymes. Molecular modeling of the HRgpA catalytic domain, based on the structure of RgpB, revealed that there are four amino acid substitutions around the active site of HRgpA relative to RgpB that may explain their different specificity. Previously, differences in the ability of these two gingipain-R forms to cleave a number of proteins were attributed to additional adhesins on HRgpA mediating increased interaction with the substrates. Here, purified RgpA(cat), the catalytic domain of HRgpA, which like RgpB also lacks adhesin subunits, was used to show that the differences between HRgpA and RgpB are probably due to the amino acid substitutions at the active site. The kinetics of cleavage of fibrinogen, a typical protein substrate for the gingipain-R enzymes, which is bound by HRgpA but not RgpA(cat) or RgpB, were evaluated, and it was shown that there was no difference in the cleavage of the fibrinogen Aalpha-chain between the different enzyme forms. HRgpA degraded the fibrinogen Bbeta-chain more efficiently, generating distinct cleavage products. This indicates that while the adhesin domain(s) play(s) a minor role in the cleavage of protein substrates, the major effect is still provided by the amino acid substitutions at the active site of rgpA gene products versus those of the rgpB gene.     

Glucuronidation of apomorphine

Apomorphine, a dopaminergic receptor agonist, is largely used in the therapy of Parkinson's disease. In this study, we characterized the glucuronidation of apomorphine and other catechols in rat liver and brain microsomes, using UDP-[U-14C]glucuronic acid and separation of the glucuronides formed by a thin layer chromatographic method. rat liver microsomes glucuronidate apomorphine at a significant rate, that was increased in the presence of dithiothreitol. Two apomorphine glucuronides were separated by high pressure liquid chromatography. We showed by electrospray mass spectrometry that both products were monoglucuronides. Other catechols were also glucuronidated in liver microsomes at various rates, and among them, 4-nitrocatechol was the most efficiently conjugated. in rat brain microsomes, only 4-nitrocatechol was significantly glucuronidated, suggesting that in the liver, several uridine-diphosphate glucuronosyltransferase (UGT) isoforms participate to the conjugation of catechols. To determine which isoforms catalyze apomorphine glucuronidation, two recombinant enzymes expressed in V79 cells were used. The isoform UGT1A6 was unable to glucuronidate apomorphine, but we observed a significant activity catalyzed by the isoform UGT2B1. These results provide, to our knowledge, the first demonstration of apomorphine conjugation by recombinant UGT2B1, and the first evidence of the lack of apomorphine glucuronidation in the rat brain.     

Enhancement of the mutagenicity of polyphenols by chlorination and nitrosation in Salmonella typhimurium.

The hydrolytic products of lignins, humic acids and industrial waste including hydroquinone, catechol, resorcinol, pyrogallol and 1,2,4-benzenetriol are widely distributed in water sources. These polyphenols can interact with chlorine or nitrite to yield new derivatives. Generally, these new products possess more mutagenic potential than their original compounds. Furthermore, the mutagenicity of these polyphenols and their derivatives can be dramatically reduced by rodent liver microsomal enzymes (S9). The mutagenicity of polyphenols is in this order: hydroquinone greater than 1,2,4-benzenetriol greater than pyrogallol, while catechol, resorcinol and phloroglucinol are non-mutagenic. The ultimate product of chlorination or nitrosation of hydroquinone has been identified to be p-benzoquinone. The formation of active oxygen species including superoxide anion and hydrogen peroxide by polyphenols has been demonstrated and this may contribute partly to the molecular mechanisms of polyphenol mutagenicity.