Catechol Metabolites Research Articles

Two-Mechanism Model for the Interaction of Etoposide Quinone with Topoisomerase IIα.

Topoisomerase II is an essential nuclear enzyme involved in regulating DNA topology to facilitate replication and cell division. Disruption of topoisomerase II function by chemotherapeutic agents is in use as an effective strategy to fight cancer. Etoposide is an anticancer therapeutic that disrupts the catalytic cycle of topoisomerase II and stabilizes enzyme-bound DNA strand breaks. Etoposide is metabolized into several species including active quinone and catechol metabolites. Our previous studies have explored some of the details of how these compounds act against topoisomerase II. In our present study, we extend those analyses by examining several effects of etoposide quinone on topoisomerase IIα including whether the quinone impacts ATP hydrolysis, DNA ligation, cleavage complex persistence, and enzyme/DNA binding. Our results demonstrate that the quinone inhibits relaxation at 100-fold lower levels of drug when compared to that of etoposide. Further, the quinone inhibits ATP hydrolysis by topoisomerase IIα. The quinone does appear to stabilize single-strand breaks similar to etoposide suggesting a traditional poisoning mechanism. However, there is minimal difference in cleavage complex persistence in the presence of etoposide or etoposide quinone. In contrast to etoposide, we find that etoposide quinone blocks enzyme/DNA binding, which provides an explanation for previous data showing the ability of the quinone to inactivate the enzyme over time. Finally, etoposide quinone is able to stabilize the N-terminal protein clamp implying an interaction between the compound and this portion of the enzyme. Taken together, the evidence supports a two-mechanism model for the effect of the quinone on topoisomerase II: (1) interfacial poison and (2) covalent poison that interacts with the N-terminal clamp and impacts the binding of DNA.

Electrochemical Label-Free Aptasensor for Specific Analysis of Dopamine in Serum in the Presence of Structurally Related Neurotransmitters

Cellular and brain metabolism of dopamine can be correlated with a number of neurodegenerative disorders, and as such, in vivo analysis of dopamine in the presence of structurally related neurotransmitters (NT) represents a holy grail of neuroscience. Interference from those NTs generally does not allow selective electroanalysis of dopamine, which redox transformation overlaps with those of other catecholamines. In our previous work, we reported an electrochemical RNA-aptamer-based biosensor for specific analysis of dopamine (Analytical Chemistry, 2013; Vol. 85, p 121). However, the overall design of the biosensor restricted its stability and impeded its operation in serum. Here, we show that specific biorecognition and electroanalysis of dopamine in serum can be performed by the RNA aptamer tethered to cysteamine-modified gold electrodes via the alkanethiol linker. The stabilized dopamine aptasensor allowed continuous 20 h amperometric analysis of dopamine in 10% serum within the physiologically important 0.1-1 μM range and in the presence of catechol and such dopamine precursors and metabolites as norepinephrine and l-DOPA. In a flow-injection mode, the aptasensor response to dopamine was ∼1 s, the sensitivity of analysis, optimized by adjusting the aptamer surface coverage, was 67 ± 1 nA μM(-1) cm(-2), and the dopamine LOD was 62 nM. The proposed design of the aptasensor, exploiting both the aptamer alkanethiol tethering to the electrode and screening of the catecholamine-aptamer electrostatic interactions, allows direct monitoring of dopamine levels in biological fluids in the presence of competitive NT and thus may be further applicable in biomedical research.

Impact of Alternaria toxins on CYP1A1 expression in different human tumor cells and relevance for genotoxicity.

The Alternaria toxins alternariol (AOH) and alternariol monomethyl ether (AME) have been reported previously to act as activators of the aryl hydrocarbon receptor (AhR) in murine hepatoma cells, thus enhancing the expression of cytochrome P450 (CYP) 1A monooxygenases. Concomitantly, both benzopyrones represent substrates of CYP1A, giving rise to catecholic metabolites. The impact of AOH and AME on CYP1A expression in human cells of different tissue origin colon (HT29), esophagus (KYSE510), liver (HepG2) and their effects on cell viability, generation of reactive oxygen species (ROS) and DNA integrity were investigated. ROS production was induced by both mycotoxins in all cell lines with AOH exhibiting the highest potency in esophageal cells concomitant with the most prominent CYP1A induction level. Of note, altertoxin-II (ATX-II), the more potent DNA-damaging mutagen formed by Alternaria alternata, induces CYP1A even at significant lower concentrations. AhR-siRNA knockdown in human esophageal cells supported the hypothesis of AhR-mediated CYP1A1 induction by AOH. However, DNA damage was minor at CYP1A1-inducing AOH concentrations. AhR-depletion did not affect the DNA-damaging properties of AOH indicating no substantial impact of AhR in this regard. However, in combination with xenobiotics prone to metabolic activation by CYP1A the induction of CYP1A by Alternaria toxins deserves further attention.

First in vitro evidence for a catechol metabolite from the anti-HIV drug efavirenz – A plausible role in toxicity

First in vitro evidence for a catechol metabolite from the anti-HIV drug efavirenz – A plausible role in toxicity

Norepinephrine and its metabolites are involved in the synthesis of neuromelanin derived from the locus coeruleus.

In order to elucidate the chemical structure of black to brown pigments, neuromelanins (NMs), in the substantia nigra (SN) and the locus coeruleus (LC) in the central nervous system of humans and other mammalian species during aging, chemical degradative methods are powerful tools. HPLC analysis after hydroiodic acid hydrolysis detected aminohydroxyphenylethylamines, aminohydroxyphenylacetic acids, and aminohydroxyethylbenzenes, which confirmed that SN‐NM and LC‐NM contain melanin derived not only from dopamine and norepinephrine (NE) but also from several other catecholic metabolites, such as 3,4‐dihydroxyphenylalanine, 3,4‐dihydroxyphenylacetic acid, 3,4‐dihydroxymandelic acid, 3,4‐dihydroxyphenylethanol, and 3,4‐dihydroxyphenylethylene glycol, in addition to the corresponding Cys‐derivatives in varying degrees. However, hydroiodic acid hydrolysis showed that LC‐NM produced the same degradation products as were detected in SN‐NM. Thus, we needed to develop a new chemical detection method to validate the existence of NE in LC‐NM. In the present study, we report that HCl hydrolysis of LC‐NM in the presence of thioglycolic acid yields new products arising from substitution of the hydroxyl group by thioglycolic acid at the benzyl position of NE and cysteinyl‐NE. This is the first chemical evidence showing that NE and cysteinyl‐NE are incorporated into LC‐NM. Using the chemical degradation methods for the determination of catechols in neuromelanin (NM), we have shown that dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), 3,4‐dihydroxyphenylethanol (DOPE), and 3,4‐dihydroxyphenylalanine (DOPA) are mainly responsible for the structure of NM from substantia nigra (SN), while norepinephrine (NE), 3,4‐dihydroxymandelic acid (DOMA), and 3,4‐dihydroxyphenylethylene glycol (DOPEG) are additionally responsible for the structure of NM from locus coeruleus (LC).

Differential Effects of Glycyrrhiza Species on Genotoxic Estrogen Metabolism: Licochalcone A Downregulates P450 1B1, whereas Isoliquiritigenin Stimulates It.

Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 μg/mL), and LigC (1 μM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 μM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 μM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 μg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women's health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.

In vitro Phase I and Phase II metabolism of α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV) and methedrone by human liver microsomes and human liver cytosol.

The aim of the present study was to identify the in vitro Phase I and Phase II metabolites of three new psychoactive substances: α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV), and methedrone, using human liver microsomes and human liver cytosol. Accurate-mass spectra of metabolites were obtained using liquid chromatography-quadrupole time-of-flight mass spectrometry. Six Phase I metabolites of α-PVP were identified, which were formed involving reduction, hydroxylation, and pyrrolidine ring opening reactions. The lactam compound was the major metabolite observed for α-PVP. Two glucuronidated metabolites of α-PVP, not reported in previous in vitro studies, were further identified. MDPV was transformed into 10 Phase I metabolites involving reduction, hydroxylation, and loss of the pyrrolidine ring. Also, six glucuronidated and two sulphated metabolites were detected. The major metabolite of MDPV was the catechol metabolite. Methedrone was transformed into five Phase I metabolites, involving N- and O-demethylation, hydroxylation, and reduction of the ketone group. Three metabolites of methedrone are reported for the first time. In addition, the contribution of individual human CYP enzymes in the formation of the detected metabolites was investigated.

Abstract 3800: Antiproliferative effect of fingolimod (FTY720) in human prostate cancer cells: Insights to estrogen metabolism in situ

Abstract Sphingokinase-1/sphingosine-1-phosphate pathway contributes significantly to progression of different types of cancer which has been recently reported to influence estrogen level in breast cancer cells. Current evidence suggests that estrogen (E2) and its metabolites, especially catechol metabolites, are playing a key role in the proliferation and carcinogenesis of the prostate glands. FTY720 is Sphingokinase-1inhibitorwhich possess anticancer properties in various tumor cell types. The aim of this study is to investigate the chemotherapeutic effect of FTYY720 against prostate cancer cells with emphasis on estrogenic microenvironment in-situ. The IC50 of FTY720 in DU145 cell line was4.2±1.4µg/ml. The concentration of estrogen and its metabolites were assayed in the media over DU145 cells using liquid chromatography tandem mass (LC/MS) technique. FTY720 treatment resulted in a dramatic decrease in estradiol (E2) and estrone (E1) levels compared to control untreated cells (2652.76±115.62 vs. 725.69±1.28 and 283.36±4.18vs. 150.32±8.96ng/g, respectively); however, it had no significant effect on the concentration of Estriol(E3). In terms of catechol estrogens, 4-hydroxyEstradiol (4-OHE2) and 2-hydroxy Estradiol (2-OHE2) formation was significantly decreased after treatment withFTY720 compared to control cells (25.42±2.40 vs. 7.45±0.82 and 485.93±3.10 vs. 477.42±4.24 ng/g), respectively. FTY720 had no significant effect on formation of 16α-hydroxyEstrone (16 α-OHE1). On the other hand, treatment with FTY720 significantly increased the concentration of 2-methoxyestrone (2-MOHE1) and 2-methoxyestradiol (2-MOHE2) compared to control cells (5.45±0.12 vs. 29.94±2.97 and165.62±5.67 vs. 269.97±15.85), respectively..The expression of catechol estrogen synthesizing enzymes (CYP19, CYP1A1 and CYP1B1) was significantly decreased within prostate cancer cells after treatment with FTY720 compared to untreated counterpart. On the other hand, the expression level of catechol estrogen degrading enzyme (COMT) was significantly decreased. Cell cycle analysis of DU145 cells showed arrest at G1 phase with significant decrease in the G2-M population due to treatment with FTY720. In conclusion, FTY720 induced anti proliferative effect in prostate cancer cells might be attributed partly to its influence on estrogen metabolism in-situ. Citation Format: Rasha M. Allam, Hisham A. Mosli, Amany E. Khalifa, Salwa M. Nofal, Ola A. Sharaf, Ashraf B. Abdel-Naim, Ahmed M. Al-Abd. Antiproliferative effect of fingolimod (FTY720) in human prostate cancer cells: Insights to estrogen metabolism in situ. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3800. doi:10.1158/1538-7445.AM2014-3800

Methylenedioxy designer drugs: Mass spectrometric characterization of their glutathione conjugates by means of liquid chromatography-high-resolution mass spectrometry/mass spectrometry and studies on their glutathionyl transferase inhibition potency

Methylenedioxy designer drugs of abuse such as 3,4-methylenedioxymethamphetamine (MDMA) can be selectively toxic to serotonergic neurons and glutathione (GSH) adducts have been implicated in its neurotoxicity. The catecholic demethylenyl metabolites of MDMA, 3,4-dihydroxymethamphetamine and 3,4-dihydroxyamphetamine, are metabolically oxidized to the corresponding ortho-quinones, which are highly reactive intermediates. These intermediates can then be conjugated with GSH preventing cellular damage. Furthermore, glutathionyl transferase (GST) activity was described to be irreversibly inhibited by the catechols dopamine, α-methyldopa and their GSH conjugates. Therefore, the aims of the present work were the detection and characterization of GSH conjugates of ten methylenedioxy drugs of abuse and their phase I metabolites as well as to assess their inhibition potency on GST activity. The substrates were incubated using human placental GST with or without preincubation by cytochrome P450 enzymes preparations. GST inhibition was tested using chlorodinitrobenzene GSH conjugation as marker reaction. GSH conjugates were analyzed and characterized using LC-high-resolution-MS/MS. For confirmation of postulated fragmentation patterns, formation of GSH conjugates of selected deuterated analogs (deuterated analogue approach, DAA) of the investigated drugs was explored. For the methylenedioxy amphetamines the following steps could be identified: conjugation of the parent compounds at position 2, 5, 6, of the demethylenyl metabolites at position 2 and 5, and of the further deaminated demethylenyl metabolites at position 2. For the β-keto-phenylalkylamine and pyrrolidinophenone, conjugation of the demethylenyl metabolites and of the deaminated demethylenyl metabolites at position 2 could be identified. The DAA allowed the differentiation of the 2 and 5/6 isomers by confirmation of the postulated mass spectral fragments. Finally, the tested drugs and phase I metabolites showed no inhibition potency on GST activity.

In vitro and in vivo biotransformation of WMS-1410, a potent GluN2B selective NMDA receptor antagonist

Structural modification of the GluN2B selective NMDA receptor antagonist ifenprodil led to the 3-benzazepine WMS-1410 with similar GluN2B affinity but higher receptor selectivity. Herein the in vitro and in vivo biotransformation of WMS-1410 is reported. Incubation of WMS-1410 with rat liver microsomes and different cofactors resulted in four hydroxylated phase I metabolites, two phase II metabolites and five combined phase I/II metabolites. With exception of catechol 4, these metabolites were also identified in the urine of a rat treated with WMS-1410. However the metabolites 7, 8 and 12 clearly show that the catechol metabolite 4 was also formed in vivo. As shown for ifenprodil the phenol of WMS-1410 represents the metabolically most reactive structural element. The biotransformation of WMS-1410 is considerably slower than the biotransformation of ifenprodil indicating a higher metabolic stability. From the viewpoint of metabolic stability the bioisosteric replacement of the phenol of WMS-1410 by a metabolically more stable moiety should be favourable.

"Ecstasy"-induced toxicity in SH-SY5Y differentiated cells: role of hyperthermia and metabolites

3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is a recreational hallucinogenic drug of abuse known to elicit neurotoxic properties. Hepatic formation of neurotoxic metabolites is thought to play a major role in MDMA-related neurotoxicity, though the mechanisms involved are still unclear. Here, we studied the neurotoxicity mechanisms and stability of MDMA and 6 of its major human metabolites, namely α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) and their correspondent glutathione (GSH) and N-acetyl-cysteine (NAC) conjugates, under normothermic (37 °C) or hyperthermic conditions (40 °C), using cultured SH-SY5Y differentiated cells. We showed that MDMA metabolites exhibited toxicity to SH-SY5Y differentiated cells, being the GSH and NAC conjugates more toxic than their catecholic precursors and MDMA. Furthermore, whereas the toxicity of the catechol metabolites was potentiated by hyperthermia, NAC-conjugated metabolites revealed higher toxicity under normothermia and GSH-conjugated metabolites-induced toxicity was temperature-independent. Moreover, a time-dependent decrease in extracellular concentration of MDMA metabolites was observed, which was potentiated by hyperthermia. The antioxidant NAC significantly protected against the neurotoxic effects of MDMA metabolites. MDMA metabolites increased intracellular glutathione levels, though depletion in thiol content was observed in MDMA-exposed cells. Finally, the neurotoxic effects induced by the MDMA metabolite N-Me-α-MeDA involved caspase 3 activation. In conclusion, this study evaluated the stability of MDMA metabolites in vitro, and demonstrated that the catechol MDMA metabolites and their GSH and NAC conjugates, rather than MDMA itself, exhibited neurotoxic actions in SH-SY5Y differentiated cells, which were differently affected by hyperthermia, thus highlighting a major role for reactive metabolites and hyperthermia in MDMA's neurotoxicity.

Etoposide Catechol Is an Oxidizable Topoisomerase II Poison

Topoisomerase II regulates DNA topology by generating transient double-stranded breaks. The anticancer drug etoposide targets topoisomerase II and is associated with the formation of secondary leukemias in patients. The quinone and catechol metabolites of etoposide may contribute to strand breaks that trigger leukemic translocations. To further analyze the characteristics of etoposide metabolites, we extend our previous analysis of etoposide quinone to the catechol. We demonstrate that the catechol is ∼2-3-fold more potent than etoposide and under oxidative reaction conditions induces high levels of double-stranded DNA cleavage. These results support a role for etoposide catechol in contributing to therapy-induced DNA damage.

Bioactivation of Sitaxentan in Liver Microsomes, Hepatocytes, and Expressed Human P450s with Characterization of the Glutathione Conjugate by Liquid Chromatography Tandem Mass Spectrometry

Sitaxentan is a selective endothelin-A receptor antagonist that was marketed as Thelin in several European countries and Canada for pulmonary arterial hypertension. Sitaxentan was undergoing further clinical trials in the United States but due to four deaths and one case of liver transplantation from severe liver toxicity that appeared to be idiosyncratic in nature, it was withdrawn worldwide in December, 2010. Sitaxentan contains a 1,3-benzodioxole ring that undergoes enzymatic demethyleneation to an ortho-catechol metabolite that can further oxidize to a reactive ortho-quinone metabolite. Here, we report the detection and mass spectral characterization of a glutathione conjugate of this sitaxentan quinone reactive metabolite that was trapped in vitro using mouse, rat, dog, and human liver microsomes supplemented with NADPH and glutathione and that was also observed in rat and human hepatocytes. Using human liver microsomes, we also demonstrated that P450 3A4 undergoes time-dependent inhibition. Density functional calculations on the catechol metabolite of sitaxentan indicated that the reaction leading to the quinone was thermodynamically favorable with an enthalpy change of -6.3 kcal/mol. Using density functional methodology, we modeled the attack of glutathione on the quinone with an S-methyl thiolate anion which allowed us to predict, based on the difference in transition state energies, that the 2-position on the phenyl ring was more likely than the 5-position as the site of glutathione conjugation. Overall, our results demonstrated that sitaxentan is capable of facile formation of a reactive ortho-quinone metabolite capable of reacting with glutathione and may rationalize the idiosyncratic nature of the hepatotoxicity that led to its withdrawal.

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.

Reactive oxygen species via redox signaling to PI3K/AKT pathway contribute to the malignant growth of 4-hydroxy estradiol-transformed mammary epithelial cells.

The purpose of this study was to investigate the effects of 17-β-estradiol (E2)-induced reactive oxygen species (ROS) on the induction of mammary tumorigenesis. We found that ROS-induced by repeated exposures to 4-hydroxy-estradiol (4-OH-E2), a predominant catechol metabolite of E2, caused transformation of normal human mammary epithelial MCF-10A cells with malignant growth in nude mice. This was evident from inhibition of estrogen-induced breast tumor formation in the xenograft model by both overexpression of catalase as well as by co-treatment with Ebselen. To understand how 4-OH-E2 induces this malignant phenotype through ROS, we investigated the effects of 4-OH-E2 on redox-sensitive signal transduction pathways. During the malignant transformation process we observed that 4-OH-E2 treatment increased AKT phosphorylation through PI3K activation. The PI3K-mediated phosphorylation of AKT in 4-OH-E2-treated cells was inhibited by ROS modifiers as well as by silencing of AKT expression. RNA interference of AKT markedly inhibited 4-OH-E2-induced in vitro tumor formation. The expression of cell cycle genes, cdc2, PRC1 and PCNA and one of transcription factors that control the expression of these genes – nuclear respiratory factor-1 (NRF-1) was significantly up-regulated during the 4-OH-E2-mediated malignant transformation process. The increased expression of these genes was inhibited by ROS modifiers as well as by silencing of AKT expression. These results indicate that 4-OH-E2-induced cell transformation may be mediated, in part, through redox-sensitive AKT signal transduction pathways by up-regulating the expression of cell cycle genes cdc2, PRC1 and PCNA, and the transcription factor – NRF-1. In summary, our study has demonstrated that: (i) 4-OH-E2 is one of the main estrogen metabolites that induce mammary tumorigenesis and (ii) ROS-mediated signaling leading to the activation of PI3K/AKT pathway plays an important role in the generation of 4-OH-E2-induced malignant phenotype of breast epithelial cells. In conclusion, ROS are important signaling molecules in the development of estrogen-induced malignant breast lesions.

Neurotoxicity of “ecstasy” and its metabolites in human dopaminergic differentiated SH-SY5Y cells

“Ecstasy” (3,4-methylenedioxymethamphetamine or MDMA) is a widely abused recreational drug, reported to produce neurotoxic effects, both in laboratory animals and in humans. MDMA metabolites can be major contributors for MDMA neurotoxicity. This work studied the neurotoxicity of MDMA and its catechol metabolites, α-methyldopamine (α-MeDA) and N-methyl-α-methyldopamine (N-Me-α-MeDA) in human dopaminergic SH-SY5Y cells differentiated with retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate.Differentiation led to SH-SY5Y neurons with higher ability to accumulate dopamine and higher resistance towards dopamine neurotoxicity. MDMA catechol metabolites were neurotoxic to SH-SY5Y neurons, leading to caspase 3-independent cell death in a concentration- and time-dependent manner. MDMA did not show a concentration- and time-dependent death. Pre-treatment with the antioxidant and glutathione precursor, N-acetylcysteine (NAC), resulted in strong protection against the MDMA metabolites’ neurotoxicity. Neither the superoxide radical scavenger, tiron, nor the inhibitor of the dopamine (DA) transporter, GBR 12909, prevented the metabolites’ toxicity.Cells exposed to α-MeDA showed an increase in intracellular glutathione (GSH) levels, which, at the 48h time-point, was not dependent in the activity increase of γ-glutamylcysteine synthetase (γ-GCS), revealing a possible transient effect. Importantly, pre-treatment with buthionine sulfoximine (BSO), an inhibitor of γ-GCS, prevented α-MeDA induced increase in GSH levels, but did not augment this metabolite cytotoxicity. Even so, BSO pre-treatment abolished NAC protective effects against α-MeDA neurotoxicity, which were, at least partially, due to GSH de novo synthesis. Inversely, pre-treatment of cells with BSO augmented N-Me-α-MeDA-induced neurotoxicity, but only slightly affected NAC neuroprotection. In conclusion, MDMA catechol metabolites promote differential toxic effects to differentiated dopaminergic human SH-SY5Y cells.

Metabolic map and bioactivation of the anti‐tumour drug noscapine

Noscapine is a promising anti-tumour agent. The purpose of the present study was to describe the metabolic map and investigate the bioactivation of noscapine. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry-based metabolomics was used to analyse the in vitro incubation mixtures, urine and faeces samples from mice treated with noscapine. Recombinant drug-metabolizing enzymes were employed to identify those involved in noscapine metabolism. Hepatic GSH levels and serum biochemistry were also carried out to determine reactive metabolites of noscapine. Several novel phase I metabolites of noscapine were detected after oral gavage of mice, including an N-demethylated metabolite, two hydroxylated metabolites, one metabolite undergoing both demethylation and cleavage of the methylenedioxy group and a bis-demethylated metabolite. Additionally, several novel glucuronides were detected, and their structures were elucidated through MS/MS fragmentology. Recombinant enzymes screening showed the involvement of several cytochromes P450, flavin-containing mono-oxygenase 1 and the UDP-glucuronosyltransferases UGT1A1, UGT1A3, UGT1A9 and UGT2B7, in noscapine metabolism. In vitro glutathione trapping revealed the existence of an ortho-quinone reactive intermediate formed through further oxidation of a catechol metabolite. However, this bioactivation process of noscapine does not occur in vivo. Similar to this result, altered glutathione levels in liver and serum biochemistry revealed no evidence of hepatic damage, thus indicating that, at least in mice, noscapine does not induce hepatotoxicity through bioactivation. A comprehensive metabolic map and bioactivation evaluation provides important information for the development of noscapine as an anti-tumour drug.

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 17β-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.

Degradation of fluorobenzene and its central metabolites 3-fluorocatechol and 2-fluoromuconate by Burkholderia fungorum FLU100

A halobenzene-degrading bacterium, Burkholderia fungorum FLU100 (DSM 23736), was isolated due to its outstanding trait to degrade fluorobenzene. Besides fluorobenzene, it utilizes, even in random mixtures, chlorobenzene, bromobenzene, iodobenzene, benzene, and toluene as sole sources of carbon and energy. FLU100 mineralizes mono-halogenated benzenes almost stoichiometrically (according to halide balance); after a lag phase, it also degrades 3-fluorophenol and 3-chlorophenol completely. The FLU100-derived transposon Tn5-mutant FLU100-P14R22 revealed 3-halocatechol to be a central metabolite of this new halobenzene degradation pathway. In FLU100, halocatechols are-as expected-strictly subject to ortho-cleavage of the catechol ring, with meta-cleavage never been observed. The strain is able to completely mineralize 3-fluorocatechol, the principal catecholic metabolite being nearly exclusively formed from fluorobenzene. The temporarily excreted 2-fluoromuconate formed thereof in turn is subsequently metabolized completely. This important finding falsifies the customary opinion of the persistence of 2-fluoromuconate valid up to now. The degradation of 4-fluorocatechol, however, being a very minor intermediate in FLU100, is substantially slower and incomplete and leads to the accumulation of uncharacterized derivatives of muconic acid and muconolactone in the medium. This branch therefore does not seem to be productive. To our knowledge, this represents the first example of the complete metabolism of 3-fluorocatechol via 2-fluoromuconate, a metabolite hitherto described as a dead-end metabolite in fluoroaromatic degradation.

Abstract 4816: Tobacco smoke modulates estrogen metabolism in the mouse lung

Abstract Tobacco smoke is the major risk factor for lung cancer; the leading cause of cancer death in both men and women in the US and worldwide. Recent studies suggest that the female hormone estrogen promotes lung cancer development. However, the relationship between tobacco smoke exposure and estrogen is not well studied. Previous data from our lab showed that whole-body exposure to tobacco smoke induced the expression of the phase I detoxification enzyme cytochrome P450 1B1 (CYP1B1) within the lungs of female A/J mice. CYP1B1 activates polyaromatic hydrocarbons in tobacco smoke and also converts estrogen to catechol metabolites, in particular 4-hydroxy estrogens (4-OHEs), which are known to be carcinogenic. The goal of this study was to characterize the profile of estrogen metabolites within the lung using a novel high-performance liquid chromatography/electrospray ionization/tandem mass spectrometry (HPLC/ESI/MS/MS) protocol. Three estrogens (E1, E2 and E3) and five estrogen metabolites (2-OHE1, 4-OHE1, 4-OHE2, 2-OMeE1 and 2-OMeE2) were detected in perfused murine lungs, with 4-OHE1 identified as the most abundant estrogen metabolite. The levels of both 4-OHE1 and 4-OHE2 were 2-fold higher within the lungs of female mice as compared to those of males. This elevation was maintained when total 4-OHE levels were normalized to total estrogen (sum of estrogen and its metabolites, p=0.009). In contrast, no gender difference was observed in 2-OHE1 levels. This estrogen metabolite profile is consistent with the results from gene expression analyses, which revealed that the ratio of CYP1B1 to CYP1A1 transcripts (CYP1A1 produces 2-OHE) was elevated (2-fold) in females as compared to males (p=0.05). To assess the impact of tobacco smoke on the profile of estrogen metabolites within the lung, female mice were exposed to tobacco smoke for 8 weeks and estrogen metabolite levels were measured by HPLC/ESI/MS/MS. As expected, the levels of 4-OHEs were significantly higher within the lungs of mice exposed to tobacco smoke (4-fold for 4-OHE1 and 2-fold for 2-OHE2) as compared to controls exposed to filtered air (p=0.00003 and 0.0007, respectively). Interestingly, the level of total 2-OMeE, a putative protective estrogen metabolite, was decreased to 70% of controls in mice exposed to tobacco smoke (p=0.005) while 2-OHE levels were comparable in control and smoke-exposed animals. These results suggest that tobacco smoke accelerates the production of carcinogenic 4-OHEs within the lung; metabolites that could potentially contribute to lung tumor development. Furthermore, CYP1B1 may represent a molecular target for therapeutic intervention in lung tumor formation. Future studies will focus on comparing the profile of estrogen metabolites in human lung tissue from never smokers and current smokers. This work was supported by funds from the Estate of Jane Villon and the Kitty Jackson Fund. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4816. doi:1538-7445.AM2012-4816