Role of the Alternaria alternata blue-light receptor LreA (white-collar 1) in spore formation and secondary metabolism.

Alternaria alternata is a filamentous fungus that causes considerable loss of crops of economically important feed and food worldwide. It produces more than 60 different secondary metabolites, among which alternariol (AOH) and altertoxin (ATX) are the most important mycotoxins. We found that mycotoxin production and spore formation are regulated by light in opposite ways. Whereas spore formation was largely decreased under light conditions, the production of AOH was stimulated 2- to 3-fold. ATX production was even strictly dependent on light. All light effects observed could be triggered by blue light, whereas red light had only a minor effect. Inhibition of spore formation by light was reversible after 1 day of incubation in the dark. We identified orthologues of genes encoding the Neurospora crassa blue-light-perceiving white-collar proteins, a cryptochrome, a phytochrome, and an opsin-related protein in the genome of A. alternata. Deletion of the white-collar 1 (WC-1) gene (lreA) resulted in derepression of spore formation in dark and in light. ATX formation was strongly induced in the dark in the lreA mutant, suggesting a repressing function of LreA, which appears to be released in the wild type after blue-light exposure. In addition, light induction of AOH formation was partially dependent on LreA, suggesting also an activating function. A. alternata ΔlreA was still able to partially respond to blue light, indicating the action of another blue-light receptor system.

Epoxide reduction to an alcohol: a novel metabolic pathway for perylene quinone-type alternaria mycotoxins in mammalian cells.

The group of perylene quinone-type Alternaria toxins contains several congeners with epoxide groups, for example, altertoxin II (ATX II) and stemphyltoxin III (STTX III). Recent studies in our laboratory have disclosed that the epoxide moieties of ATX II and STTX III are reduced to alcohols in human colon Caco-2 cells, thereby resulting in the formation of altertoxin I (ATX I) and alteichin, respectively. In the present study, this pathway was demonstrated for ATX II in three other mammalian cell lines. Furthermore, the chemical reaction of this toxin with monothiols like glutathione could be shown, and the structures of the reaction products were tentatively elucidated by UV and mass spectrometry. Chemical reaction of ATX II with dithiols capable of forming five- and six-membered rings gave rise to ATX I, thus providing a clue for the molecular mechanism of the epoxide reduction pathway of ATX II. Both epoxide reduction and glutathione conjugation appear to attenuate, but not completely abolish, the genotoxicity of ATX II.

Catechol formation: a novel pathway in the metabolism of sterigmatocystin and 11-methoxysterigmatocystin.

The mycotoxin sterigmatocystin (STC) has an aflatoxin-like structure including a furofuran ring system. Like aflatoxin B1, STC is a liver carcinogen and forms DNA adducts after metabolic activation to an epoxide at the furofuran ring. In incubations of STC with human P450 isoforms, one monooxygenated and one dioxygenated STC metabolite were recently reported, and a GSH adduct was formed when GSH was added to the incubations. However, the chemical structures of these metabolites were not unambiguously elucidated. We now report that hepatic microsomes from humans and rats predominantly form the catechol 9-hydroxy-STC via hydroxylation of the aromatic ring. No STC-1,2-oxide and only small amounts of STC-1,2-dihydrodiol were detected in microsomal incubations, suggesting that epoxidation is a minor pathway compared to catechol formation. Catechol formation was also much more pronounced than furofuran epoxidation in the microsomal metabolism of 11-methoxysterigmatocystin (MSTC). In support of the preference of catechol formation, only trace amounts of the thiol adduct of the 1,2-oxides but large amounts of the thiol adducts of the 9-hydroxy-8,9-quinones were obtained when N-acetyl-l-cysteine was added to the microsomal incubations of STC and MSTC. In addition to hydroxylation at C-9, smaller amounts of 12c-hydroxylated, 9,12c-dihydroxylated, and 9,11-dihydroxylated metabolites were formed. Our study suggests that hydroxylation of the aromatic ring, yielding a catechol, represents a major and novel pathway in the oxidative metabolism of STC and MSTC, which may contribute to the toxic and genotoxic effects of these mycotoxins.

DNA reactivity of altertoxin II: Identification of two covalent guanine adducts formed under cell-free conditions.

Fungi of the genus Alternaria infest many agricultural crops and produce numerous mycotoxins, of which altertoxin II (ATX II) is one of the most mutagenic metabolites. ATX II carries an epoxide group but the formation of DNA adducts has not been demonstrated to date. We report now that ATX II gives rise to two covalent adducts with guanine when incubated with DNA under cell-free conditions. These adducts were demonstrated by LC-high resolution MS after enzymatic degradation of the incubated DNA to deoxynucleosides. The major adduct results from the covalent binding of ATX II, presumably through the epoxide group, to guanine, whereas the minor guanine adduct is derived from the major one by the elimination of two equivalents of water. In addition, a third adduct was detected, formed through covalent binding of ATX II to cytosine followed by the loss of two equivalents of water. The direct DNA reactivity of ATX II may explain its high mutagenicity.

Novel oxidative metabolites of the mycoestrogen zearalenone in vitro.

The estrogenic mycotoxin zearalenone (ZEN) is known to get metabolized to the alpha-and beta-isomers of zearalenol, but no hydroxylation products of ZEN have yet been reported as metabolites in animals or humans. We have therefore incubated ZEN with microsomes from rat liver in the presence of a nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-regenerating system and analyzed the extracted metabolites with HPLC and GC-MS after trimethylsilylation. A total of 17 in vitro metabolites were observed. The two major metabolites were tentatively identified as monohydroxylated ZEN with the newly introduced hydroxyl group localized in the aliphatic macrocyclic ring. According to the GC-MS analysis, other six monohydroxylation products of ZEN were formed as minor metabolites, together with alpha-and beta-zearalenol and monohydroxylated zearalenols. Thus, ZEN has a considerable propensity for undergoing metabolic hydroxylation reactions in vitro, and the in vivo formation and biological properties of such oxidative metabolites should now be studied.

Glucuronidation of curcuminoids by human microsomal and recombinant UDP-glucuronosyltransferases.

Glucuronidation is an important pathway in the metabolism of curcumin, but the isoforms of uridine-5'-diphosphoglucuronosyltransferase (UGT) involved are not known. Here, we report on the glucuronidation of the three natural curcuminoids and their major phase I metabolites with microsomes from human liver and intestine as well as with human recombinant UGTs. Microsomes from human liver generated predominantly the phenolic and small amounts of the alcoholic glucuronide of each curcuminoid, whereas intestinal microsomes formed only the phenolic conjugates but with higher activities. The phenolic glucuronidation of the curcuminoids was predominantly catalyzed by hepatic UGT1A1 and intestinal UGT1A8 and 1A10, whereas UGT1A9, 2B7, and 1A8 exhibited high activities for hexahydro-curcuminoids. UGT1A9 was able to form the alcoholic glucuronide of each curcuminoid in addition to the phenolic conjugate. These data suggest that the gastrointestinal tract contributes substantially to the glucuronidation of curcuminoids in humans, which may have important implications for their pharmacokinetic fate in vivo.

Novel oxidative in vitro metabolites of the mycotoxins alternariol and alternariol methyl ether.

The Alternaria toxins alternariol (AOH; 3,7,9-trihydroxy-1-methyl-6H-benzo[c]chromen-6-one) and alternariol methyl ether (AME, 3,7-dihydroxy-9-methoxy-1-methyl-6H-benzo[c]chromen-6-one) are common contaminants of food and feed, but their oxidative metabolism in mammals is as yet unknown. We have therefore incubated AME and AOH with microsomes from rat, human, and porcine liver and analyzed the microsomal metabolites with HPLC and GC-MS/MS. Seven oxidative metabolites of AME and five of AOH were detected. Their chemical structures were derived from their mass spectra using deuterated trimethylsilyl (TMS) derivatives, and from the information obtained from enzymatic methylation. Several of the metabolites were identified by comparison with synthetic reference compounds. AME as well as AOH were monohydroxylated at each of the four possible aromatic carbon atoms and also at the methyl group. In addition, AME was demethylated to AOH and dihydroxylated to a small extent. As the four metabolites arising through aromatic hydroxylation of AME and AOH are either catechols or hydroquinones, the oxidative metabolism of these mycotoxins may be of toxicological significance.

Curcuminoids form reactive glucuronides in vitro.

Curcumin is of current interest because of its putative anti-inflammatory, anticarcinogenic, and anti-Alzheimer's activity, but its pharmacokinetic and metabolic fate is poorly understood. The present in vitro study has therefore been conducted on the glucuronidation of curcumin and its major phase I metabolite, hexahydro-curcumin, as well as of various natural and artificial analogs. The predominant glucuronide generated by rat and human liver microsomes from curcumin, hexahydro-curcumin, and other analogs with a phenolic hydroxyl group was a phenolic glucuronide according to LC-MS/MS analysis. However, a second glucuronide carrying the glucuronic acid moiety at the alcoholic hydroxyl group was formed from the same curcuminoids, but not hexahydro-curcuminoids, by human microsomes. Curcuminoids without a phenolic hydroxyl group gave rise to the aliphatic glucuronide only. The phenolic glucuronides of curcuminoids, but not of hexahydro-curcuminoids, were rather lipophilic and, in part, unstable in aqueous solution, their stability depending strongly on the type of aromatic substitution. The phenolic glucuronide of curcumin and of its natural congeners, but not the parent compounds, clearly inhibited the assembly of microtubule proteins under cell-free conditions, implying chemical reactivity of the glucuronides. These novel properties of the major phase II metabolites of curcuminoids deserve further investigation.

Ochratoxin A: apoptosis and aberrant exit from mitosis due to perturbation of microtubule dynamics?

Ochratoxin A (OTA) is a potent nephrotoxin and causes high incidences of renal tumors in rodents. The molecular events leading to tumor formation by OTA are not well defined. Early pathological changes observed in kidneys of rats treated with OTA in vivo include frequent mitotic and abnormally enlarged cells, detachment of tubule cells, and apoptosis within the S3 segment of the proximal tubule, suggesting that OTA may interfere with molecules involved in the regulation of cell division and apoptosis. In this study, treatment of immortalized human kidney epithelial (IHKE) cells with OTA (0-50 microM) resulted in a time- and dose-dependent increase in apoptosis and activation of c-Jun N-terminal kinase. At the same time, OTA blocked metaphase/anaphase transition and led to the formation of aberrant mitotic figures and giant cells with abnormally enlarged and/or multiple nuclei, sometimes still connected by chromatin bridges. Immunostaining of the mitotic apparatus using an alpha-tubulin antibody revealed defects in spindle formation. In addition, OTA inhibited microtubule assembly in a concentration-dependent manner in a cell-free, in vitro assay. Interestingly, treatment with OTA also resulted in activation of the transcription factor nuclear factor kappa B (NFkappaB), which has recently been shown to promote cell survival during mitotic cell cycle arrest. Based on these observations, we hypothesize that the mechanism by which OTA promotes tumor formation involves interference with microtubuli dynamics and mitotic spindle formation, resulting in apoptosis or-in the presence of survival signals such as stimulation of the NFkappaB pathway-premature exit from mitosis. Aberrant exit from mitosis resulting in blocked or asymmetric cell division may favor the occurrence of cytogenetic abnormalities and may therefore play a critical role in renal tumor formation by OTA.

Stimulation of estradiol glucuronidation: a protective mechanism against estradiol-mediated carcinogenesis?

17beta-Estradiol (E2) and its catechol and methoxy metabolites are believed to play important roles in the mechanism of E2-mediated tumor formation. Because conjugation with glucuronic acid lowers tissue levels by facilitating excretion, we have determined the kinetic parameters of the glucuronidation of E2, estrone (E1), and seven phase I metabolites using human liver microsomes. The catechol estrogens 2- and 4-hydroxy-E2/E1 exhibited the highest clearance, exceeding that of E2, E1, and the methoxy metabolites by factors of 6-44. Homotropic activation kinetics were observed for the 3-glucuronidation of E2 but not for any of the metabolites. None of the metabolites affected the kinetics of the 3-glucuronidation of E2. In contrast, the isoflavone daidzein stimulated the formation of E2-3-glucuronide, as has been reported previously. This heterotropic activation by daidzein appears to be specific for the glucuronidation of E2 because daidzein did not affect the glucuronidation of the 2- and 4-hydroxy metabolites of E2. However, daidzein may lower the glucuronidation of 2-methoxy-E2 in vivo due to its preferential glucuronidation. The decreased tissue levels of E2 and increased concentrations of 2-methoxy-E2, as implied by this study and the previous one, may contribute to the protective effect of daidzein against breast and endometrial cancer.

Microsomal hydroxylation and glucuronidation of [6]-gingerol.

[6]-Gingerol is the major pungent principle of ginger and frequently is ingested with various condiments and nutritional supplements. We report here that incubation of [6]-gingerol with NADPH-fortified rat hepatic microsomes gave rise to eight metabolites, which were tentatively identified by GC-MS analysis as two products of aromatic hydroxylation as well as the diastereomers of two aliphatic hydroxylation products and the diastereomers of [6]-gingerdiol. Hepatic microsomes from rats and humans fortified with UDPGA glucuronidated [6]-gingerol predominantly at the phenolic hydroxyl group, but small amounts of a second monoglucuronide involving the aliphatic hydroxyl group were also identified by LC-MS/MS analysis. Human intestinal microsomes formed the phenolic glucuronide only. Supersomes containing human UGT1A1 and 1A3 exclusively generated the phenolic glucuronide, albeit with very low activities, whereas UGT1A9 catalyzed the specific formation of the alcoholic glucuronide and UGT2B7 the predominant formation of the phenolic glucuronide with high activities. Our study indicates a rather complex metabolism of [6]-gingerol, which should be taken into consideration for the multiple biological activities of this compound.

Metabolism of curcuminoids in tissue slices and subcellular fractions from rat liver.

Curcumin and its natural congeners are of current interest because of their putative anti-inflammatory and anticarcinogenic activities, but knowledge about their metabolic fate is scant. In the present study conducted with precision-cut liver slices from male and female Sprague-Dawley rats, five reductive but no oxidative metabolites of curcumin and its demethoxy and bis-demethoxy analogues were observed and identified by HPLC and GC-MS analysis, mostly by comparison with authentic reference compounds. The major reductive metabolites were the hexahydrocurcuminoids in both male and female rat liver slices, whereas male rats formed more octahydro than tetrahydro metabolites and female rats more tetrahydro- than octahydrocurcuminoids. Tetrahydro, hexahydro, and octahydro metabolites were predominantly present as glucuronides, but a significant proportion of sulfate conjugates was also observed. The lack of formation of oxidative metabolites of curcumin and the ready generation of reductive metabolites were confirmed using rat liver microsomes and cytosol, respectively. Results of enzymatic hydrolysis studies conducted under various conditions revealed that curcumin and demethoxycurcumin are chemically less stable than bis-demethoxycurcumin, whereas the reductive metabolites of all three curcuminoids are stable compounds. This is the first report on the metabolism of demethoxycurcumin and bis-demethoxycurcumin. In view of the chemical instability of the parent curcuminoids, it is proposed to use their major phase I metabolites, that is, the stable hexahydro products, as biomarkers for exposure in clinical studies.

Sulfoglucosides as Novel Modified Forms of the Mycotoxins Alternariol and Alternariol Monomethyl Ether.

The mycotoxins alternariol and alternariol-9-O-methyl ether have recently been reported to be extensively conjugated with glucose and malonyl glucose in tobacco suspension cells. However, only trace amounts of glucosylated conjugates were detected in tomatoes inoculated with Alternaria alternata in the present study. Instead, mostly sulfate conjugates were observed. In studies using cultures of A. alternata and incubations of alternariol and alternariol-9-O-methyl ether with tomato tissue in the absence of the fungus, it was clarified that sulfate conjugates were produced by the fungus, whereas tomato tissues converted alternariol and alternariol-9-O-methyl ether to glucosylated metabolites. Alternariol-3-sulfate, alternariol-9-sulfate, and alternariol-9-O-methyl ether-3-sulfate were unambiguously identified as fungal metabolites using MS and 1H and 13C NMR spectroscopy. When these sulfate conjugates were incubated with tobacco suspension cells or ex planta tomato tissues, three sulfoglucosides of alternariol and one sulfoglucoside of alternariol-9-O-methyl ether were formed. Using NMR spectroscopy, the chemical structures of alternariol-3-sulfate-9-glucoside, alternariol-9-sulfate-3-glucoside, and alternariol-9-O-methyl ether-3-sulfate-7-glucoside were established. These conjugates were also detected in the A. alternata-inoculated tomato. This is the first report on a mixed sulfate/glucoside diconjugate of a mycotoxin. Diconjugates of this novel type may be formed by all mycotoxins and their phase I metabolites with two or more hydroxyl groups and should be taken into account in the future analysis of modified mycotoxins.

DNA damage and repair kinetics of the Alternaria mycotoxins alternariol, altertoxin II and stemphyltoxin III in cultured cells.

The Alternaria mycotoxins alternariol (AOH) and altertoxin II (ATX II) have previously been shown to elicit mutagenic and genotoxic effects in bacterial and mammalian cells, although with vastly different activities. For example, ATX II was about 50 times more mutagenic than AOH. We now report that stemphyltoxin III (STTX III) is also highly mutagenic. The more pronounced effects of the perylene quinones ATX II and STTX III at lower concentrations compared to the dibenzo-α-pyrone AOH indicate a marked dependence of the genotoxic potential on the chemical structure and furthermore suggest that the underlying modes of action may be different. We have now further investigated the type of DNA damage induced by AOH, ATX II and STTX III, as well as the repair kinetics and their dependence on the status of nucleotide excision repair (NER). DNA double strand breaks induced by AOH due to poisoning of topoisomerase IIα were completely repaired in less than 2h. Under cell-free conditions, inhibition of topoisomerase IIα could also be measured for ATX II and STTX III at low concentrations, but the perylene quinones were catalytic inhibitors rather than topoisomerase poisons and did not induce DSBs. DNA strand breaks induced by ATX II and STTX III were more persistent and not completely repaired within 24h. A dependence of the repair rate on the NER status could only be demonstrated for STTX III, resulting in an accumulation of DNA damage in NER-deficient cells. Together with the finding that the DNA glycosylase formamidopyrimidine-DNA glycosylase (Fpg), but not T4 endonuclease V, is able to generate additional DNA strand breaks measurable by the alkaline unwinding assay, we conclude that the genotoxicity of the perylene quinones with an epoxide group is probably caused by the formation of DNA adducts which may be converted to Fpg sensitive sites.

Patulin reduces glutathione level and enzyme activities in rat liver slices.

In the present study, an attempt was made to identify glutathione (GSH) adducts of patulin in precision-cut rat liver slices, which were used as a model system to study the metabolism and biological effects of this mycotoxin. Patulin disappeared in the slices but none of the GSH adducts, previously demonstrated in the chemical reaction of patulin with GSH, could be detected by HPLC. After incubation with various concentrations of patulin, a concentration-dependent decline of the GSH level was observed in the slices. For example, only 25% of the GSH of controls was found with 200 microM patulin. The activities of glutathione-S-transferase (GST) and of drug metabolizing phase I and phase II enzymes, assayed by the hydroxylation and conjugation of testosterone, were also reduced. On the other hand, incubation with patulin markedly increased lipid peroxidation in the slices. The effects of patulin on enzyme activities and lipid peroxidation may be a consequence of the GSH decline, which cannot be accounted for by a direct reaction of patulin with GSH due to the high concentration of GSH in hepatocytes. The decrease of GSH level and GST activity may be related to the putative mutagenic and carcinogenic potential of patulin.

Conjugation of the mycotoxins alternariol and alternariol monomethyl ether in tobacco suspension cells.

The mycotoxins alternariol (AOH) and alternariol-9-O-methyl ether (AME) carry three and two phenolic hydroxyl groups, respectively, which makes them candidates for the formation of conjugated metabolites in plants. Such conjugates may escape routine methods of analysis and have therefore been termed masked or, more recently, modified mycotoxins. We report now that AOH and AME are extensively conjugated in suspension cultures of tobacco BY-2 cells. Five conjugates of AOH were identified by MS and NMR spectroscopy as ß-D-glucopyranosides (attached in AOH 3- or 9-position) as well as their 6'-malonyl derivatives, and as a gentiobiose conjugate. For AME, conjugation resulted in the d-glucopyranoside (mostly attached in the AME 3-position) and its 6'- and 4'-malonyl derivatives. Pronounced differences were noted for the quantitative pattern of AOH and AME conjugates as well as for their phytotoxicity. Our in vitro study demonstrates for the first time that masked mycotoxins of AOH and AME can be formed in plant cells.

Permeation and metabolism of Alternaria mycotoxins with perylene quinone structure in cultured Caco-2 cells.

The absorption of four Alternaria toxins with perylene quinone structures, i.e. altertoxin (ATX) I and II, alteichin (ALTCH) and stemphyltoxin (STTX) III, has been determined in the Caco-2 cell Transwell system, which represents a widely accepted in vitro model for human intestinal absorption and metabolism. The cells were incubated with the four mycotoxins on the apical side, and the concentration of the toxins in the incubation media of both chambers and in the cell lysate were determined by liquid chromatography coupled with diode array detection and mass spectrometry (LC-DAD-MS) analysis. ATX I and ALTCH were not metabolised in Caco-2 cells, but ATX II and STTX III were partly biotransformed by reductive de-epoxidation to the metabolites ATX I and ALTCH, respectively. Based on the apparent permeability coefficients (Papp), the following ranking order for the permeation into the basolateral compartment was obtained: ATX I > ALTCH >> ATX II > STTX III. Total recovery of the four toxins decreased in the same order. It is assumed that the losses of STTX III, ATX II and ALTCH in Caco-2 cells are caused by covalent binding to cell components due to the epoxide group and/or the α,ß-unsaturated carbonyl group present in these toxins. We conclude from this study that ATX I and ALTCH are well absorbed from the intestinal lumen into the portal blood in vivo. For ATX II and STTX III, intestinal absorption of the parent toxins is very low, but these toxins are partly metabolised to ATX I and ALTCH, respectively, in the intestinal epithelium and absorbed as such.

Curcumin uptake and metabolism.

Curcumin (CUR) is the major orange pigment of turmeric and believed to exert beneficial health effects in the gastrointestinal tract and numerous other organs after oral intake. However, an increasing number of animal and clinical studies show that the concentrations of CUR in blood plasma, urine, and peripheral tissues, if at all detectable, are extremely low even after large doses. The evidence and possible reasons for the very poor systemic bioavailablity of CUR after oral administration are discussed in this brief review. Major factors are the chemical instability of CUR at neutral and slightly alkaline pH, its susceptibility to autoxidation, its avid reductive and conjugative metabolism, and its poor permeation from the intestinal lumen to the portal blood. In view of the very low intestinal bioavailablity, it is difficult to attribute the putative effects observed in peripheral organs to CUR. Therefore, metabolites and/or degradation products of CUR should be taken into consideration as mediators of the pharmacological activity.

Metabolism and permeability of curcumin in cultured Caco-2 cells.

SCOPE: Curcumin (CUR) and its major metabolite hexahydro-CUR were studied in Caco-2 cells and in the Caco-2 Millicell® system in vitro to simulate their in vivo intestinal metabolism and absorption in humans. METHODS AND RESULTS: Analysis of the incubation medium and cell lysate showed that Caco-2 cells reduce CUR to hexahydro-CUR and octahydro-CUR, and conjugate CUR and its reductive metabolites with glucuronic acid and sulfate. Using the Caco-2 Millicell® system, an efficient transfer of the conjugates into the basolateral, but not the apical, compartment was observed after apical administration. Likewise, hexahydro-CUR was reduced to octahydro-CUR, and glucuronide and sulfate conjugates almost exclusively permeated to the basolateral side. The apparent permeability coefficients (Papp values) of CUR, hexahydro-CUR and their metabolites were determined and found to be extremely low for unchanged CUR, but somewhat higher for hexahydro-CUR and the conjugated metabolites. CONCLUSION: The results of this study clearly show that the systemic bioavailability of CUR from the intestine after oral intake must be expected to be virtually zero. Reductive and conjugated metabolites, formed from CUR in the intestine, exhibit moderate absorption. Thus, any biological effects elicited by CUR in tissues other than the gastrointestinal tract are likely due to CUR metabolites.

Catechol metabolites of the mycotoxin zearalenone are poor substrates but potent inhibitors of catechol-O-methyltransferase.

The mycotoxin zearalenone (ZEN) elicits estrogenic effects and is biotransformed to two catechol metabolites, in analogy to the endogenous steroidal estrogen 17ß-estradiol (E2). Previous studies have shown that the catechol metabolites of ZEN have about the same potency to induce oxidative DNA damage as the catechol metabolites of E2, but are less efficiently converted to their methyl ethers by human hepatic catechol-O-methyltransferase (COMT). Here, we report that the two catechol metabolites of ZEN, i.e. 13-hydroxy-ZEN and 15-hydroxy-ZEN, are not only poor substrates of human COMT but are also able to strongly inhibit the O-methylation of 2-hydroxy-E2, the major catechol metabolite of E2. 15-Hydroxy-ZEN acts as a non-competitive inhibitor and is about ten times more potent than 13-hydroxy-ZEN, which is an uncompetitive inhibitor of COMT. The catechol metabolites of ZEN were also shown to inhibit the O-methylation of 2-hydroxy-E2 by hepatic COMT from mouse, rat, steer and piglet, although to a lesser extent than observed with human COMT. The powerful inhibitory effect of catechol metabolites of ZEN on COMT may have implications for the tumorigenic activity of E2, because catechol metabolites of E2 elicit genotoxic effects, and their impaired O-methylation may increase the tumorigenicity of steroidal estrogens.