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.

Local fixation of antibiotics with fibrin spray on soft tissues: experimental study on the relevance of the application techniques.

OBJECTIVE: In acute and chronic infections of soft tissue, a radical surgical debridement is necessary, and is regarded as the 'Gold Standard, as well as an immediately systemic antibiotic therapy. Additional treatment with local antibiotics and/or antibiotic-containing materials is commonly used in clinical settings, but the efficient concentration of the antibiotic is not well-known. METHODS: Aside from the typical procedures which are provided and supported by the industry, we have conducted an animal study including 39 Sprague Dawley rats. We have treated the back sores with local antibiotics (vancomycin) alone or in two combinations with a fibrin-glue spray technique. RESULTS: It could be demonstrated that, in particular, the simultaneous application of vancomycin and fibrin leads to a stable antibiotic concentration and a long period of time wherein the antibiotic substance remains in the tissue. CONCLUSIONS: This easy and inexpensive method of application can be a promising factor for the clinical improvement of local antibiotic therapy.

A Data Science-Based Analysis Points at Distinct Patterns of Lipid Mediator Plasma Concentrations in Patients With Dementia.

Based on accumulating evidence of a role of lipid signaling in many physiological and pathophysiological processes including psychiatric diseases, the present data driven analysis was designed to gather information needed to develop a prospective biomarker, using a targeted lipidomics approach covering different lipid mediators. Using unsupervised methods of data structure detection, implemented as hierarchal clustering, emergent self-organizing maps of neuronal networks, and principal component analysis, a cluster structure was found in the input data space comprising plasma concentrations of d = 35 different lipid-markers of various classes acquired in n = 94 subjects with the clinical diagnoses depression, bipolar disorder, ADHD, dementia, or in healthy controls. The structure separated patients with dementia from the other clinical groups, indicating that dementia is associated with a distinct lipid mediator plasma concentrations pattern possibly providing a basis for a future biomarker. This hypothesis was subsequently assessed using supervised machine-learning methods, implemented as random forests or principal component analysis followed by computed ABC analysis used for feature selection, and as random forests, k-nearest neighbors, support vector machines, multilayer perceptron, and naïve Bayesian classifiers to estimate whether the selected lipid mediators provide sufficient information that the diagnosis of dementia can be established at a higher accuracy than by guessing. This succeeded using a set of d = 7 markers comprising GluCerC16:0, Cer24:0, Cer20:0, Cer16:0, Cer24:1, C16 sphinganine, and LacCerC16:0, at an accuracy of 77%. By contrast, using random lipid markers reduced the diagnostic accuracy to values of 65% or less, whereas training the algorithms with randomly permuted data was followed by complete failure to diagnose dementia, emphasizing that the selected lipid mediators were display a particular pattern in this disease possibly qualifying as biomarkers.

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women II: Total estrogenicity calculations accounting for competitive protein and receptor binding and potency.

Evaluating the biological significance of human-relevant exposures to environmental estrogens involves assessing the individual and total estrogenicity of endogenous and exogenous estrogens found in serum, for example from biomonitoring studies. We developed a method for this assessment by integrating approaches for (i) measuring total hormone concentrations by mass spectrometry (Fleck et al., 2018), (ii) calculating hormone bioavailable concentrations in serum and, (iii) solving multiple equilibria between estrogenic ligands and receptors, and (iv) quantitatively describing key elements of estrogen potency. The approach was applied to endogenous (E1, E2, E3, E4), environmental (BPA), and dietary Genistein (GEN), Daidzein (DDZ) estrogens measured in the serum of thirty pregnant women. Fractional receptor occupancy (FRO) based estrogenicity was dominated by E1, E2 and E3 (ER-α, 94.4-99.2% (median: 97.3%), ER-ß, 82.7-97.7% (median: 92.8%), as was the total response (TR), which included ligand specific differences in recruitment of co-activator proteins (RCA). The median FRO for BPA was at least five orders of magnitude lower than E1, E2 and E3, and three orders of magnitude lower than the fetal derived E4 and GEN and DDZ. BPA contributed less than 1/1000th of the normal daily variability in total serum estrogenicity in this cohort of pregnant women.

Inhibition of the protein kinase IKKepsilon attenuates neuropathic pain in mice.

Inhibitor-kappaB kinase epsilon (IKKε, Ikbke) constitutes an NF-κB activating kinase with high homology to the classical I-κB kinase subunits, IKKα and IKKß. It is expressed in nociceptive neurons in the spinal cord and in dorsal root ganglia (DRG) and involved in inflammatory nociception. Under inflammatory conditions, IKKε deficient mice show significantly less nociceptive behavior in comparison to wild type mice associated with reduced activation of NF-κB and attenuated NF-κB-dependent gene expression. The role of IKKε in neuropathic pain has not been investigated so far. We applied the spared nerve injury (SNI) model of neuropathic pain in mice and found an increased expression of IKKε in the spinal cord, the DRGs and the sciatic nerve after induction of neuropathy. Genetic depletion of IKKε or pharmacological inhibition by amlexanox led to a significant reduction of mechanical hyperalgesia and cold allodynia in comparison to control mice. Transcription factor ELISA indicated that the effects are mediated by reduced activation of NF-κB. Furthermore, immunofluorescence staining, qPCR and Western Blot analyses revealed that the decreased pain-like behavior was associated with a reduced activation of microglia, diminished expression of c-fos as well as a decreased activation of MAP-Kinases. In summary, we conclude that IKKε modulates mechanisms of neuropathic pain by activating NF-κB. The administration of IKKε inhibitors might therefore constitute a new and promising approach for the therapy of neuropathic pain.

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women I: Serum levels, variability and the basis for urinary biomonitoring of serum estrogenicity.

Biomonitoring of human exposure to estrogens most frequently focuses on environmental and dietary estrogens, and infrequently includes measures of exposure to potent endogenous estrogens present in serum. Pregnancy is a developmentally sensitive period during which "added" serum estrogenicity exceeding normal intra-individual daily variability may be of particular relevance. We made repeated measurements of serum concentrations of estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), daidzein (DDZ), genistein (GEN) and bisphenol A (BPA) in thirty pregnant women using ultra-performance liquid chromatography coupled with tandem mass spectrometry detection (UPLC-MS/MS) and electrospray ionization (ESI). Serum E1, E2, and E3 concentrations varied significantly (coefficients of variation 9-10%) with broad ranges across the cohort: 1.61-85.1 nM, 9.09-69.7 nM, and 1.5-36.3 nM respectively. BPA (undetected, estimated from total exposure), DDZ and GEN concentrations were 1-5 orders of magnitude lower. The 24-h urinary elimination profiles of endogenous estrogens were each strongly correlated with their corresponding serum concentrations (Pearson's Correlation Coefficients of 0.83 (E1), 0.84 (E2) and 0.94 (E3)). A multivariate regression analysis produced equations for estimating serum concentrations of E1, E2, E3, E4, GEN and DDZ from urinary elimination rates and gestation period, an important step towards non-invasive biomonitoring for assessment of "added" estrogenicity during pregnancy.

Metabolism and pharmacokinetics of zearalenone following oral and intravenous administration in juvenile female pigs.

Zearalenone (ZEN) is a well-studied mycotoxin whose potent estrogenic properties have been used by international regulatory bodies to set health-based guidance values for ZEN exposure in grain-based foods from changes in hormonally responsive tissues of juvenile female pigs. The role of metabolism in determining estrogenic responses in vivo is a major uncertainty in inter-species extrapolation to humans and in assessing the potential for added susceptibility in sensitive subpopulations. This study evaluated the metabolism of ZEN and pharmacokinetics in ∼2 month-old female pigs using oral and intravenous dosing. The absolute bioavailability (AUCoral/AUCIV) of receptor-active ZEN aglycone was 1.8 ± 0.80%, consistent with extensive pre-systemic Phase II conjugation. Reductive metabolism to α-zearalenol (α-ZEL) was extensive, with smaller amounts of ß-ZEL. When combined with its higher binding affinity, relative to ZEN and ß-ZEL, α-ZEL was the predominant contributor to total estrogen receptor ligand activity (∼90%) after oral dosing with ZEN. The apparent similarities of reductive and Phase II conjugation metabolism of ZEN between pigs and humans support the use of juvenile female pigs as a sensitive model for risk assessments of estrogenic effects from dietary ZEN.

Urine and serum biomonitoring of exposure to environmental estrogens II: Soy isoflavones and zearalenone in pregnant women.

UNLABELLED: Urine and serum biomonitoring was used to measure internal exposure to selected dietary estrogens in a cohort of 30 pregnant women. Exposure was measured over a period comprising one-half day in the field (6 h) and one day in a clinic (24 h). Biomonitoring of the dietary phytoestrogens genistein (GEN), daidzein (DDZ) and equol (EQ), as well as the mycoestrogen, zearalenone (ZEN) and its congeners, was conducted using UPLC-MS/MS. Biomonitoring revealed evidence of internal exposure to naturally occurring dietary estrogens during pregnancy. Urinary concentrations of total GEN, DDZ and EQ were similar to levels reported for general adult U.S. POPULATION: Measurable concentrations of total (parent and metabolites) GEN, DDZ and EQ were present in 240, 207 and 2 of 270 serum samples, respectively. Six out of 30 subjects had measurable concentrations of unconjugated GEN and/or DDZ in serum between 0.6 and 7.1 nM. Urine to serum total isoflavone ratios for GEN, DDZ and EQ were 13, 47, and 180, respectively. ZEN and its reductive metabolite, α-zearalenol (α-ZEL), were present in pregnant women (11 out of 30 subjects) as conjugates at levels near the limit of quantification. The average total urinary concentration was 0.10 µg/L for ZEN and 0.11 µg/L for α-ZEL.

Pharmacokinetics of isoflavones from soy infant formula in neonatal and adult rhesus monkeys.

Consumption of soy infant formula represents a unique exposure scenario in which developing children ingest a mixture of endocrine-active isoflavones along with a substantial portion of daily nutrition. Genistein and daidzein were administered as glucoside conjugates to neonatal rhesus monkeys in a fortified commercial soy formula at 5, 35, and 70 days after birth. A single gavage dosing with 10 mg/kg bw genistein and 6 mg/kg bw daidzein was chosen to represent the upper range of typical daily consumption and to facilitate complete pharmacokinetic measurements for aglycone and total isoflavones and equol. Adult monkeys were also gavaged with the same formula solution at 2.8 and 1.6 mg/kg bw genistein and daidzein, respectively, and by IV injection with isoflavone aglycones (5.2 and 3.2 mg/kg bw, respectively) to determine absolute bioavailability. Significant differences in internal exposure were observed between neonatal and adult monkeys, with higher values for dose-adjusted AUC and Cmax of the active aglycone isoflavones in neonates. The magnitude and frequency of equol production by the gut microbiome were also significantly greater in adults. These findings are consistent with immaturity of metabolic and/or physiological systems in developing non-human primates that reduces total clearance of soy isoflavones from the body.

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.

Isoflavones in soy flour diet have different effects on whole-genome expression patterns than purified isoflavone mix in human MCF-7 breast tumors in ovariectomized athymic nude mice.

SCOPE: Soy flour diet (MS) prevented isoflavones from stimulating MCF-7 tumor growth in athymic nude mice, indicating that other bioactive compounds in soy can negate the estrogenic properties of isoflavones. The underlying signal transduction pathways to explain the protective effects of soy flour consumption were studied here. METHODS AND RESULTS: Ovariectomized athymic nude mice inoculated with MCF-7 human breast cancer cells were fed either Soy flour diet (MS) or purified isoflavone mix diet (MI), both with equivalent amounts of genistein. Positive controls received estradiol pellets and negative controls received sham pellets. GeneChip Human Genome U133 Plus 2.0 Array platform was used to evaluate gene expressions, and results were analyzed using bioinformatics approaches. Tumors in MS-fed mice exhibited higher expression of tumor growth suppressing genes ATP2A3 and BLNK and lower expression of oncogene MYC. Tumors in MI-fed mice expressed a higher level of oncogene MYB and a lower level of MHC-I and MHC-II, allowing tumor cells to escape immunosurveillance. MS-induced gene expression alterations were predictive of prolonged survival among estrogen-receptor-positive breast cancer patients, whilst MI-induced gene changes were predictive of shortened survival. CONCLUSION: Our findings suggest that dietary soy flour affects gene expression differently than purified isoflavones, which may explain why soy foods prevent isoflavones-induced stimulation of MCF-7 tumor growth in athymic nude mice.

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.

Determination of the absolute configuration of perylene quinone-derived mycotoxins by measurement and calculation of electronic circular dichroism spectra and specific rotations.

Altertoxins I-III, alterlosins I and II, alteichin (alterperylenol), stemphyltoxins I-IV, stemphyperylenol, stemphytriol, 7-epi-8-hydroxyaltertoxin I, and 6-epi-stemphytriol are mycotoxins derived from perylene quinone, for which the absolute configuration was not known. Electronic circular dichroism (ECD) spectra were calculated for these compounds and compared with measured spectra of altertoxins I-III, alteichin, and stemphyltoxin III and with reported Cotton effects. Specific rotations were calculated and compared with reported specific rotations. The absolute configuration of all the toxins, except for stemphyltoxin IV, could thus be determined. The validity of the assignment was high whenever reported ECD data were available for comparison, and the validity was lower when the assignment was based only on the comparison of calculated and reported specific rotations. ECD spectra are intrinsically different for toxins with a biphenyl substructure and for toxins derived from dihydroanthracene.

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.

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.

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.