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.

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.

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.

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.

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.

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.

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.

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.

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.

Alternaria toxins: Altertoxin II is a much stronger mutagen and DNA strand breaking mycotoxin than alternariol and its methyl ether in cultured mammalian cells.

Altertoxin II (ATX II) is one of the several mycotoxins produced by Alternaria fungi. It has a perylene quinone structure and is highly mutagenic in Ames Salmonella typhimurium, but its mutagenicity in mammalian cells has not been studied before. Here we report that ATX II is a potent mutagen in cultured Chinese hamster V79 cells, inducing a concentration-dependent increase of mutations at the hypoxanthine guanine phosphoribosyltransferase gene locus at concentrations similar to that of the established mutagen 4-quinoline-N-oxide. Thus, ATX II is at least 50-times more potent as a mutagen than the common Alternaria toxins alternariol (AOH) and alternariol methyl ether (AME). In contrast to AOH and AME, ATX II does not affect the cell cycle of V79 cells. ATX II also causes DNA strand breaks in V79 cells, with a potency again exceeding that of AOH and AME. The high mutagenic and DNA strand breaking activity of ATX II raises the question of whether this Alternaria toxin poses a risk for public health, and warrants studies on the occurrence of ATX II and other perylene quinone-type mycotoxins in food and feed.

Catechol metabolites of zeranol and 17ß-estradiol: a comparative in vitro study on the induction of oxidative DNA damage and methylation by catechol-O-methyltransferase.

α-Zearalanol (α-ZAL, zeranol) is a highly estrogenic macrocyclic ß-resorcylic acid lactone, which is used as a growth promotor for cattle in various countries. We have recently reported that α-ZAL and its major metabolite zearalanone (ZAN) are hydroxylated at the aromatic ring by microsomes from human liver in vitro, thereby forming two catechol metabolites each. Thus, the oxidative metabolism of α-ZAL and ZAN resembles that of the endogenous steroidal estrogens 17ß-estradiol (E2) and estrone (E1), which also give rise to two catechols each. As these catechol metabolites are believed to mediate the carcinogenicity of E2 and E1 by causing oxidative DNA damage and DNA adducts, their methylation by catechol-O-methyltransferase (COMT) is an important inactivation pathway. Here we report that hepatic microsomes from five species generate catechol metabolites of α-ZAL and ZAN, the highest amounts being formed by human liver microsomes, followed by rat, mouse, steer and swine. The microsomal extracts and the individual catechols of α-ZAL, ZAN, E2 and E1 were found to induce oxidative DNA damage, as measured by the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in a cell-free system. The ranking of pro-oxidant activity was 15-HO-ZAN>15-HO-α-ZAL≈4-HO-E2/E1≈2-HO-E2/E1>13-HO-ZAN>13-HO-α-ZAL. With respect to the rate of methylation by human hepatic COMT, the ranking was 2-HO-E2/E1>>4-HO-E2/E1>15-HO-α-ZAL/ZAN>>13-HO-α-ZAL/ZAN. Thus, some catechol metabolites of α-ZAL and ZAN are better pro-oxidants and poorer substrates of COMT than the catechols of E2 and E1. These findings warrant further investigations into the genotoxic potential of α-ZAL, which may constitute another biological activity in addition to its well-known estrogenicity.

Hydroxylation of the mycotoxin zearalenone at aliphatic positions: novel mammalian metabolites.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species and frequently found as a contaminant of food and feed. Earlier studies have disclosed that ZEN is biotransformed in microsomes from human and rat liver to multiple hydroxylated metabolites, two of which have recently been identified as products of aromatic hydroxylation. Here, we report for the first time on the structure elucidation of metabolites arising through hydroxylation of the aliphatic ring of ZEN at various positions. By using reference compounds and ZEN labeled with deuterium at specific positions, evidence was provided for the preferential hydroxylation of ZEN at C-8 and, to a lesser extent, at C-9, C-10, and C-5. In contrast, hydroxylation at C-6 could be ruled out, as could oxidation of the olefinic double bond. These results imply that the phase I metabolism of ZEN in the mammalian organism is more extensive than previously thought, and warrant further studies on the in vivo formation of the novel ZEN metabolites and their biological activities.

Genotoxicity and inactivation of catechol metabolites of the mycotoxin zearalenone.

Zearalenone (ZEN) is a highly estrogenic mycotoxin produced by Fusarium species. The adverse effects of ZEN and its reductive metabolite α-zearalenol (α-ZEL) are often compared to those of 17ß-estradiol (E2) and estrone (E1). These endogenous steroidal estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (1) hormonal activity and (2) genotoxic effects after cytochrome P450-catalyzed metabolic activation to catechols. Like E1 and E2, ZEN and α-ZEL exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites. The subsequent methylation of catechols by catechol-O-methyltransferase (COMT) is generally considered as a detoxifying pathway. Imbalances between the activation and inactivation reactions can lead to the formation of reactive semiquinones and quinones, which can alkylate DNA or produce reactive oxygen species by redox cycling. In the present study, the genotoxicity of the catechol metabolites of ZEN, α-ZEL, E1 and E2 was determined in a cell-free system by measuring 8-oxo-2'-deoxyguanosine using a LC-DAD-MS(2) method. Each of the individual catechols of ZEN, α-ZEL, E1 and E2 induced oxidative DNA damage in calf thymus DNA. The ranking order of the DNA damaging activity was 15-hydroxy-ZEN/α-ZEL ≈ 2/4-hydroxy-E1/E2 > 13-hydroxy-ZEN/α-ZEL. When hepatic microsomes from different species were incubated with ZEN, the rat had the highest activity for catechol formation, followed by human, mouse, pig and steer. The amount of catechol metabolites correlated directly with the amount of oxidative damage in calf thymus DNA. The ranking order for the rate of methylation by human hepatic COMT was 2-hydroxy-E1/E2 >> 4-hydroxy-E1/E2 >> 13/15-hydroxy-ZEN/α-ZEL. Thus, the catechol metabolites of the mycoestrogen ZEN and its reductive metabolite α-ZEL exhibit a DNA-damaging potential comparable to that of the catechol metabolites of E1 and E2, but are much poorer substrates for inactivation by human COMT.

Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro.

SCOPE: Zearalenone (ZEN) and α-zearalanol (α-ZAL, zeranol) were studied in differentiated Caco-2 cells and in the Caco-2 Millicell® system in vitro to simulate their in vivo intestinal absorption and metabolism in humans. METHODS AND RESULTS: In addition to metabolic reduction/oxidation, extensive conjugation with glucuronic acid and sulfate of the parent compounds and their phase I metabolites was observed. The positional isomers of the glucuronides and sulfates were unambiguously identified: Sulfonation occurred specifically at the 14-hydroxyl group, whereas glucuronidation was less specific and, in addition to the preferred 14-hydroxyl group, involved the 16- and 7-hydroxyl groups. Using the Caco-2 Millicell® system, an efficient transfer of the glucuronides and sulfates of ZEN and α-ZAL and their phase I metabolites into both the basolateral and the apical compartment was observed after apical administration. The apparent permeability coefficients (P(app) values) of ZEN, α-ZAL and the ZEN metabolite α-zearalenol were determined, using an initial apical concentration of 20 µM and a permeation time of 1 h. CONCLUSION: According to the P(app) values, the three compounds are expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycones and as glucuronide and sulfate conjugates in humans.

Oxidative metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in precision-cut rat liver slices in vitro.

SCOPE: Monohydroxylation of alternariol (AOH) and alternariol-9-methyl ether (AME) has previously been reported as a prominent metabolic route under cell-free conditions. This pathway gives rise to several catechol metabolites and may therefore be of toxicological relevance. METHODS AND RESULTS: To clarify whether hydroxylation of AOH and AME occurs under in vivo-like conditions in the presence of conjugation reactions, the metabolism of the Alternaria toxins has now been studied in precision-cut rat liver slices. Four catechol metabolites of AOH and two of AME, together with several of their O-methylation products, as catalyzed by catechol-O-methyl transferase, were clearly identified after incubation of the liver slices with AOH and AME. These metabolites were predominantly present as conjugates with glucuronic acid and/or sulfate. In preliminary studies with bile duct-cannulated male rats dosed with AOH by gavage, the four monohydroxylated metabolites of AOH could also be demonstrated in the bile either as catechols or as O-methyl ethers. CONCLUSION: These experiments clearly show that AOH and AME undergo catechol formation in vivo and warrant closer examination of the toxicological significance of this metabolic pathway.

Identification of an aliphatic epoxide and the corresponding dihydrodiol as novel congeners of zearalenone in cultures of Fusarium graminearum.

The mycotoxin zearalenone (ZEN) is produced by various Fusarium fungi and frequently found as a contaminant in food and feed. There are reports in the literature that several closely related analogues of ZEN are also formed in cultures of Fusarium species. We have therefore analyzed the organic extract from a 40 day culture of Fusarium graminearum by LC-DAD-MS and detected 15 compounds, which could be congeners of ZEN because of their ultraviolet, mass spectroscopy, and tandem mass spectroscopy spectra. In addition to confirming the previously reported α- and ß-stereoisomers of 5-hydroxy-ZEN and 10-hydroxy-ZEN, we identified seven ZEN congeners for the first time. One of the major novel congeners was shown by nuclear magnetic resonance spectroscopy and chemical synthesis to have the structure of an aliphatic ZEN epoxide, whereas two minor products proved to be the corresponding dihydrodiols. In addition, three stereoisomers of a cyclization product of the dihydrodiols, carrying a spiro-acetal group, were identified as fungal products for the first time. The latter may be artifacts, because the ZEN epoxide and dihydrodiol are unstable under acidic conditions and rearrange easily to the spiro-acetal compounds.

Glucuronidation of zearalenone, zeranol and four metabolites in vitro: formation of glucuronides by various microsomes and human UDP-glucuronosyltransferase isoforms.

Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α-zearalanol (α-ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α-ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP-glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14-hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α-ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.

Aromatic hydroxylation and catechol formation: a novel metabolic pathway of the growth promotor zeranol.

Alpha-zearalanol (alpha-ZAL, zeranol) is a macrocyclic resorcylic acid lactone, which is highly estrogenic and used as a growth promotor for cattle in various countries. Little is known about the phase I metabolism of alpha-ZAL. We now report that alpha-ZAL and its major metabolite zearalanone (ZAN) are extensively monohydroxylated at the aromatic ring by microsomes from human liver in vitro. This novel pathway leads to catechols, the chemical structures of which were unambiguously established by the use of deuterium-labeled alpha-ZAL and ZAN, and by the synthesis of authentic standards. The aromatic hydroxylation of alpha-ZAL is almost exclusively mediated by the human cytochrome P450 (hCYP) 1A2 isoform. The catechol metabolites of alpha-ZAL and ZAN are unstable and readily oxidized to quinones, which could be detected among the metabolites of alpha-ZAL and ZAN generated by human hepatic microsomes and hCYP1A2. Furthermore, the quinone metabolites are able to form covalent adducts with N-acetylcysteine (NAC), as several of such adducts were found in microsomal incubations fortified with NAC. Aromatic hydroxylation of alpha-ZAL was also observed with bovine, porcine and rat hepatic microsomes. Further studies are needed to demonstrate the catechol pathway of alpha-ZAL in vivo and to assess its toxicological significance.