Impact of phase I metabolism on uptake, oxidative stress and genotoxicity of the emerging mycotoxin alternariol and its monomethyl ether in esophageal cells
- 1.1k Downloads
Studies on the genotoxicity of Alternaria mycotoxins focus primarily on the native compounds. Alternariol (AOH) and its methyl ether (AME) have been reported to represent substrates for cytochrome P450 enzymes, generating hydroxylated metabolites. The impact of these phase I metabolites on genotoxicity remains unknown. In the present study, the synthesis and the toxicological effects of the metabolites 4-hydroxy alternariol (4-OH-AOH) and 4-hydroxy alternariol monomethyl ether (4-OH-AME) are presented and compared to the effects of the parent molecules. Although the two phase I metabolites contain a catecholic structure, which is expected to be involved in redox cycling, only 4-OH-AOH increased reactive oxygen species (ROS) in human esophageal cells (KYSE510), 4 times more pronounced than AOH. No ROS induction was observed for 4-OH-AME, although the parent compound showed some minor impact. Under cell-free conditions, both metabolites inhibited topoisomerase II activity comparable to their parent compounds. In KYSE510 cells, both metabolites were found to enhance the level of transient DNA–topoisomerase complexes in the ICE assay. Although the level of ROS was significantly increased by 4-OH-AOH, neither DNA strand breaks nor enhanced levels of formamidopyrimidine-DNA-glycosylase (FPG)-sensitive sites were observed. In contrast, AOH induced significant DNA damage in KYSE510 cells. Less pronounced or even absent effects of hydroxylated metabolites compared to the parent compounds might at least partly be explained by their poor cellular uptake. Glucuronidation as well as sulfation appear to have only a minor influence. Instead, methylation of 4-OH-AOH seems to be the preferred way of metabolism in KYSE510 cells, whereby the toxicological relevance of the methylation product remains to be clarified.
KeywordsAlternaria alternata Reactive oxygen species Topoisomerase inhibition KYSE510 Human phase I and II metabolism Mycotoxin conjugates
Pfeiffer et al. (2007b, 2008) incubated human microsomes with AOH and AME confirming the formation of metabolites hydroxylated at C-2, C-4 and C-8. Furthermore, CYP1A1, commonly occurring in extrahepatic tissues such as the esophagus (Lechevrel et al. 1999), was the most active monooxygenase for AOH and especially for AME (Pfeiffer et al. 2008; Schreck et al. 2012). Thus, phase I metabolites may be generated in a tissue-specific manner after ingestion of AOH or AME and may at least contribute to the induction of esophageal cancer (Liu et al. 1991). CYP1A1 belongs to the isoenzyme family of CYPs which is mainly regulated by the aryl hydrocarbon receptor (AhR) pathway. As hypothesized by Schreck et al. (2012), AOH and AME are inducers of the AhR pathway, which enhances the expression of several metabolizing enzymes especially CYP monooxygenases. Experiments with murine AhR-deficient Hepa-1c1c12 cells did not show induction of CYP expression after incubation with AOH or AME supporting the hypothesis. Also in line are the findings of Pahlke et al. (2015), who analyzed the impact of Alternaria toxins on CYP1A transcription and activity in different human tumor cells with the objective to identify potential organ specificity. AOH caused an induction of CYP1A most prominently in esophageal cells (KYSE510) after 24-h incubation, whereas AME only mediated an increase in liver cells. Because of the enhanced sensitivity of KYSE510 cells toward AOH, the experiments were repeated in AhR-suppressed KYSE510 cells. CYP1A1 induction by AOH was significantly reduced compared to the AhR-expressing cells, but AhR suppression was of no relevance for the DNA-damaging properties of AOH. The data suggest that at least AOH promotes its xenobiotic metabolism by AhR-dependent induction of CYP enzymes in cells.
The lacking impact of AhR suppression on DNA damage might be due to the initiation of cellular defense mechanisms. As recently reported, AME and, to a greater extent, AOH were found to modulate the cellular redox status in human colon cancer cells (HT29), human liver cancer cells (HepG2) and especially KYSE510 cells (Pahlke et al. 2015; Tiessen et al. 2013a). The increase in reactive oxygen species (ROS) results from an imbalance between ROS formation and elimination by scavengers and might on the one hand affect directly DNA, proteins or lipids and on the other hand act as signaling molecules e.g., in the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. Nrf2 is bound by Kelch-like ECH-associated protein 1 (Keap1) in the cytosolic fraction (Itoh et al. 1999). Electrophiles and ROS provoke the release of the Nrf2 from the Nrf2–Keap1 complex, which can now translocate into the nucleus where it binds in a complex with small Maf proteins to the antioxidant response element (ARE) and initiates the transcription of anti-oxidative enzymes like glutathione-S-transferases (GST), γ-glutamylcysteine synthetases (γ-GCL) or UDP-glucuronosyltransferases (UGT). Furthermore, Miao et al. (2005) reported a direct regulation of Nrf2 by AhR activation leading to an elaborated drug-metabolizing detoxification mechanism made up of phase I and II enzymes. As stated above, the catecholic structure of the phase I metabolites may contribute to the induction of ROS. It can therefore be hypothesized that biotransformation of AOH and AME plays an important role in the severity of the toxic effects. Until now, little is known about the impact of possible metabolites of AOH and AME. It entails the necessity of further investigation of the phase I metabolites to better assess the health risk of Alternaria toxins.
In the present study, the question was addressed whether phase I metabolites of AOH and AME with their highly reactive catechol or hydroquinone structure might exceed the toxicological effects of their parent compounds. In a previous study, KYSE510 cells reacted most sensitive toward AOH regarding ROS production and CYP1A induction compared to HepG2 or HT29 cells (Pahlke et al. 2015). Based on these results and a potential link between a high incidence of esophageal cancer in a province of China and the consumption of grains heavily contaminated with Alternaria (Liu et al. 1991, 1992), esophageal tumor cells were chosen in this study. In order to compare the effects of the metabolites with those of the parent toxins, the impact on cytotoxicity, induction of ROS generation, topoisomerase targeting, genotoxicity and cellular uptake were investigated in detail.
Materials and methods
Chemicals and reagents
AOH and AME were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). 4-OH-AOH and 4-OH-AME were chemically synthesized as shown in Fig. 1. Experimental details are described in the Online Resource 1. The products were purified by semi-preparative HPLC (Knauer, Germany), characterized by NMR spectroscopy (Bruker Avance UltraShield 400, Ettlingen, Germany) and HRMS analysis (Thermo Scientific LTQ Orbitrap XL Hybrid FTMS, Waltham, MA, USA), and their purity (>95 %) was analyzed by HPLC-DAD (Agilent 1200, Waldbronn, Germany) at a wavelength of 340 nm. Etoposide (ETO) was bought from Sigma-Aldrich (Taufkirchen, Germany). Kinetoplast DNA (kDNA) and topoisomerase IIα were obtained from TopoGEN\ (Port Orange, FL, USA). Topoisomerase IIα and IIβ-specific rabbit polyclonal antibodies, specific rabbit polyclonal IgG antibody (sc-101762) and goat anti-rabbit IgG-HRP antibody (sc-2004) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The DNA repair enzyme formamidopyrimidine-DNA-glycosylase (FPG) from Escherichia coli was obtained from New England Biolabs (Frankfurt am Main, Germany). Sulfatase (from Aerobacter aerogenes, Type VI) and β-glucuronidase (from E. coli) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). All other chemicals and reagents were purchased from Carl Roth GmbH Co. KG (Karlsruhe, Germany) or Sigma-Aldrich Chemie GmbH unless stated otherwise.
The human esophageal carcinoma cell line KYSE510 was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany). Culture media and supplements were purchased from GIBCO Invitrogen (Karlsruhe, Germany). KYSE510 cells were cultivated in Rosswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin (50 units/mL and 50 µg/mL, respectively). The Chinese hamster ovary cell line CHO-ARE-luciferase as described in Heiss et al. (Heiss et al. 2014) was cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10 % FCS, 4 µg/mL puromycin and 1 % l-glutamine. Both cell lines grew in humidified incubators at 37 °C and 5 % CO2. Cells were routinely tested for the absence of mycoplasma contamination.
Cytotoxicity assay (WST-1)
Mitochondrial activity as a measure of cytotoxicity was determined in KYSE510 esophageal carcinoma cells with the WST-1 test kit (Roche Applied Science, Mannheim, Germany). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium is reduced to a water-soluble formazan salt by mitochondrial enzymes of the cells. The formazan salt can be detected photometrical. 1,300 KYSE510 cells/well were seeded in 96-well plates and cultivated for 48 h. Thereafter, cells were incubated with AOH and 4-OH-AOH or the positive control Triton X-100 for 24 h in serum-containing medium. The assay was performed according to the manufacturer’s protocol, and absorbance was measured at 450 nm with a reference wavelength of 650 nm with a plate reader (Victor3 V, Perkin Elmer, Waltham, MA, USA). Cell viability was specified as mitochondrial activity and calculated as treated cells to control cells × 100 (% T/C).
Dichlorofluorescein (DCF) assay
The production of cellular ROS, indicative for increased oxidative stress, was measured by the DCF assay as reported in Keston and Brandt (1965). The assay is based on the uptake of the non-fluorescent dihydrodichlorofluorescein diacetate and intracellular de-esterification to dihydrodichlorofluorescein, which is oxidized to 2′,7′-dichlorofluorescein by reactive oxygen species. DCF can be measured fluorometrically at an excitation of 485 nm with the multimode plate reader Victor3 V (Perking Elmer, Waltham, MA, USA). KYSE510 cells were seeded 48 h prior to incubation in a black 96-well plate. Cells were incubated with AOH, AME, 4-OH-AOH, 4-OH-AME or menadione (MEN), used as positive control compound, in colorless serum-free medium for 1 h. Oxidative stress was determined as relative fluorescence and calculated as treated cells over control cells (oxidative stress (%) = 100 × (emission of treated sample)/(emission of control).
Nrf2 reporter gene assay
The ARE-dependent luciferase assay was performed as described in Heiss et al. (2014). Transfected CHO cells were plated at a density of 6 × 105 cells/well in 96-well plates and cultivated for 4 h in serum-containing medium. Subsequently, cells were incubated with AOH, AME, 4-OH-AOH, 4-OH-AME (solvent vehicle 0.5 % DMSO) or the positive control 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO; 100 nM) for 20 h in serum-containing medium. After the incubation, the cells were washed with PBS twice and frozen at least for 1 h at −80 °C. Then 50 µL luciferase lysis buffer (Promega; E1531) was added to each well and shaken for 10 min. 40 µL of the cell lysate was transferred into a black 96-well plate. The fluorescence signal of GFP was measured at 485-nm excitation and 520-nm emission wavelength, and the ATP and luciferin solution were added and measured afterward automatically by the Genios Pro plate reader (Tecan, Grödig, Austria). The ratio of chemiluminescence/GFP fluorescence was formed and normalized to solvent control (Nrf2 activation = (ratio of treated sample)/(ratio of solvent control).
Single cell gel electrophoresis (comet assay)
Single cell gel electrophoresis was performed according to Tice et al. (2000). A total of 300,000 KYSE510 cells were spread into Petri dishes (Ø 5.5 cm) and allowed to grow for 48 h. Subsequently, cells were treated with AOH, AME, 4-OH-AOH and 4-OH-AME for 1 h in serum-free medium. UV-B light (λ = 312 nm; dosage: 448 J/cm2) was used as a positive control. Subsequent working steps were done according to Pelka et al. (2009). Additional treatment was performed with the DNA repair enzyme FPG from E. coli (New England Biolabs, Vienna, Austria) according to the manufacturer’s protocol, using 2 × 0.08 units FPG for 30 min at 37 °C. This allows the detection of additional oxidative damage to DNA bases (Hatahet et al. 1994; Tchou et al. 1994). Fluorescence microscopy was performed with a Zeiss Axioskop 40 FL (λex = 546 ± 12 nm; λem ≥ 590 nm) after staining with ethidium bromide. Slides were subjected to computer-aided image analysis (Comet Assay IV System, Perceptive Instruments, Suffolk, UK), scoring 2 × 50 randomly picked cells per slide. The results were parameterized with respect to intensity of DNA in the comet tail and calculated as percentage of overall DNA intensity in the respective cell.
The cell-free decatenation assay was used to determine the catalytic activity of topoisomerase IIα to release DNA minicircles from kDNA. A mixture of topoisomerase IIα (TopoGEN) and supercoiled kDNA (200 ng) was incubated for 60 min at 37 °C with the respective AOH, AME, 4-OH-AOH and 4-OH-AME concentrations according to the manufacturer’s protocol with slight modifications (TopoGEN, Inc., Florida, USA). ETO (50 µM) served as a positive control. To stop the reaction, 5 µL loading buffer was added and samples were electrophoresed in 1 % agarose gel. The gel was dyed with ethidium bromide (10 µg/mL in distilled water) and documented by digital photography under UV light using the LAS 4000 (Fujifilm, Tokyo, Japan).
In-vivo-complex-of-enzyme (ICE) assay
Briefly, 3 × 106 KYSE510 cells were seeded in Petri dishes (Ø 15 cm) for 72 h. Afterward, cells were incubated with AOH, AME, 4-OH-AOH, 4-OH-AME or 50 µM of the topoisomerase poison ETO as a positive control for 1 h in serum-free medium. The cell lysate was layered on a cesium chloride gradient and centrifuged at 100,000×g for 22 h at 20 °C. 300 µL fractions of each sample were collected. The fractions were blotted on a nitrocellulose membrane, and the topoisomerase complexes were conjugated with topoisomerase IIβ-specific rabbit polyclonal antibodies (Santa Cruz Biotechnology). An anti-rabbit IgG peroxidase conjugate was used as secondary antibody. The respective chemiluminescent signals (Lumi-GLO, Cell Signaling Technology, Danvers, USA) were analyzed using the LAS 4000 (Fujifilm, Tokyo, Japan). Arbitrary light units were referred to the DNA content of the fractions and plotted as treated sample over control × 100 (%, T/C).
Cellular uptake and metabolism of AOH, AME, 4-OH-AOH and 4-OH-AME (LC-MS/MS)
Optimized ESI-MS and ESI-MS/MS parameters, retention times and LOD/LOQ values of the applied LC-ESI-MS/MS method
Precursor ion (m/z)
Product ionsa (m/z)
Preparation of calibration solutions and spiking procedure
Solid reference standards were dissolved to a concentration of 1 mg/mL in methanol. From the 1 mg/mL standard, dilution series were created in methanol to yield concentrations of 1, 5, 10, 50, 100, 150, 200 ng/mL. These standards were utilized to generate a calibration curve for external calibration purpose. To evaluate the extraction efficiency and the matrix effects preliminary spiking experiments were performed. One concentration of each toxin (2.5 µM) was spiked to the scratched cells or the medium in triplicate before the extraction to cover the whole sample preparation procedure. Before analysis, the spiked samples were diluted 1:10. Quantitative data evaluation was performed using the Xcalibur Quan Browser software.
For statistical evaluation, the Origin software was used. All results represent the mean of at least 3 independent experiments ± standard deviation (SD). Concentration-dependent data were statistically analyzed as stated in the figure legends.
Impact of AOH and 4-OH-AOH on mitochondrial activity
AOH, AME and 4-OH-AOH influence the intracellular redox status
Activation of the ARE-dependent Nrf2 pathway by AOH, 4-OH-AOH and AME
Inhibition of topoisomerase IIα activity by AOH, AME, 4-OH-AOH and 4-OH-AME in a cell-free system
Previous studies demonstrated that AOH interferes with human topoisomerases, an effect which is likely to at least contribute to the genotoxic properties of the compound. The impact of 4-hydroxylation on the activity of human topoisomerase II was investigated in the decatenation assay under cell-free conditions, exemplified for the topoisomerase IIα isoform using etoposide (ETO, 50 µM) as a positive control. Active topoisomerase II releases minicircles from the complex high molecular kinetoplast DNA network (Fig. 6a, lane 10, 6b, lane 11), thus enabling the released minicircles to migrate in the agarose gel (Fig. 6a, b, lane 1). Inhibition of topoisomerase II activity was already apparent at a concentration of 10 µM 4-OH-AOH, and the enzyme activity was completely suppressed with 25 µM 4-OH-AOH (Fig. 6a, lane 8) as evident by the blunted release of free minicircles from the catenated kDNA by topoisomerase IIα. In contrast, AOH showed the first significant sign of suppression at 50 µM (lane 5), even though the line of free DNA minicircles vanishes already at 10 µM (lane 3). A significant difference between AOH and 4-OH-AOH was present at a concentration of 25 µM. Thus, under cell-free conditions, the inhibitory potential of the phase I metabolite 4-OH-AOH was more intense than for the parent compound. 10 µM AME inhibited the catalytic activity of topoisomerase IIα significantly (Fig. 6b, lane 3). A complete blockage of the enzyme was apparent at 25 µM 4-OH-AME (Fig. 6b, lane 8), but there was no statistically significant difference between the metabolite and AME. Taken together, under cell-free conditions, AOH appears as a less potent inhibitor of topoisomerase IIα compared to AME and both 4-OH-metabolites.
Topoisomerase II poisoning by AOH, AME and the oxidative metabolites in KYSE510 cells
To verify topoisomerase inhibition on the cellular level, the ICE assay was used, determining the amount of cleavable complexes formed with DNA and topoisomerase IIα or IIβ in KYSE510 after 1-h incubation with 10 µM and 50 µM AOH, AME, 4-OH-AOH and 4-OH-AME. No topoisomerase IIα–DNA complexes were detected for any of the test substances (data not shown). Topoisomerase IIβ–DNA complexes were significantly raised to 277 ± 107 % by AOH, 208 ± 61 % by 4-OH-AOH, 203 ± 38 % by AME and 143 ± 36 % by 4-OH-AME with the highest concentration tested (Fig. 7). Yet, no statistically significant differences were found between the different test compounds.
DNA-damaging potential of AOH, AME and their 4-hydroxy metabolites
Interference with topoisomerases as well as oxidative stress may cause DNA damage. The effects of AOH, AME and their 4-hydroxy metabolites on DNA integrity in KYSE510 cells were determined in the comet assay after 1-h incubation. Formamidopyrimidine-DNA-glycosylase (FPG) was included in the comet assay protocol to detect FPG-sensitive sites as an indication for oxidative DNA damage. A significant raise in tail intensities (5 ± 3.5 %, FPG-treated 6.6 ± 1.4 %) mediated by 50 µM AOH was detected, whereas 4-OH-AOH (50 µM; 3.1 ± 0.4 %, FPG-treated 3.7 ± 2.9 %) did not significantly affect DNA integrity. In contrast to AOH, neither significant increased DNA strand break levels nor additional oxidative DNA lesions after FPG treatment were observed for AME and 4-OH-AME.
Cellular uptake of AOH, AME and the hydroxylated metabolites
Considering the substantial discrepancy between the effects of the hydroxylated metabolites on topoisomerase II under cell-free conditions and the impact on DNA integrity in the comet assay, the question was addressed whether differences in cellular uptake between the compounds might have to be considered. Cellular uptake of AOH, AME and their 4-hydroxylated metabolites was determined after 1-h incubation by analyzing the cell culture medium and the cell lysate by LC-MS/MS. A typical LC-MS/MS chromatogram is illustrated in Online Resource 2 (Supplement Fig. S1). In the cell lysate, the average amount of AOH was 304 ± 127 ng/mg protein and for AME 885 ± 269 ng/mg protein (Fig. 8a). The concentration of 4-OH-AOH (2 ± 1 ng/mg protein) and 4-OH-AME (13 ± 1 ng/mg protein) in the cell lysate was substantially lower compared to the parent substances. The apparent recovery in the cell lysate matrix was 78 ± 1 % for AOH, 60 ± 7 % for AME as well as 59 ± 10 and 73 ± 10 % for 4-OH-AOH and 4-OH-AME, respectively. No significant difference could be observed in the cell lysates after glucuronidase and sulfatase treatment. Neither newly formed hydroxylated products of AOH or AME nor a degeneration of 4-OH-AOH and 4-OH-AME to AOH and AME, respectively, were found in the medium or the cell lysate after 1-h incubation time. Instead, incubations with 4-OH-AOH showed an additional peak in the chromatogram eluting after 3.6 min (see Online Resource 3; Supplement Fig. S2). A compound with an m/z of 287 and the same SRMs as 4-OH-AME was observed.
In the cell culture medium 5 ± 2.5 µM AOH (apparent recovery 95 ± 28 %), 1.9 ± 0.5 µM AME (apparent recovery 104 ± 20 %), 1.3 ± 0.2 µM 4-OH-AOH (apparent recovery 44 ± 12 %) and 0.4 µM 4-OH-AME (apparent recovery 63 ± 18 %) were detected after 1-h incubation (Fig. 8b). Moreover, a significant difference in the analyzed culture medium of the 4-OH-AOH incubations was detected after sulfatase treatment when compared to the standard phosphate buffer samples. Also an increase in 4-OH-AOH in the medium after glucuronidase incubation was apparent but not yet significant. The other three substances revealed no differences between the standard incubation and the glucuronidase and sulfatase treatment (Fig. 8b).
Alternaria toxins belong to the group of so-called emerging mycotoxins, being not regulated so far, but with more and more data demonstrating their frequency of occurrence in feed and food and their genotoxic properties. Previous studies already indicated under in vitro conditions the formation of oxidative metabolites of AOH and AME and the induction of oxidative stress (Burkhardt et al. 2011; Pahlke et al. 2015; Pfeiffer et al. 2007b). The present study investigated and compared the effects of AOH and AME to the phase I metabolites 4-OH-AOH and 4-OH-AME in human esophageal cancer cells with respect to the induction of oxidative stress, the ability to target topoisomerase II as a central genotoxic mechanism of AOH and the consequences for DNA integrity under consideration of cellular uptake and metabolism.
The fluorescence signal in the DCF assay, indicative for enhanced intracellular ROS levels in KYSE510 cells mediated by AOH (Fig. 3), was in accordance with previous data on the induction of oxidative stress by AOH in different cell lines (Fernández-Blanco et al. 2014; Pahlke et al. 2015; Solhaug et al. 2012; Tiessen et al. 2013a). In KYSE510 cells, AME was significantly less potent than AOH to enhance the intracellular ROS level, indicating that the methyl-moiety at C9-position might play a role. Of note, the cellular uptake of AME in KYSE510 cells was almost two times higher than for AOH (Fig. 8a), potentially due to the higher lipophilicity of AME. These data are contrary to the findings of Burkhardt et al. (2009) who reported a faster uptake for AOH than for AME in human colorectal adenocarcinoma cells (Caco2) after 1-h incubation and a concentration of 20 µM. These results argue for the fact that the toxicokinetics of AOH and AME are cell-type specific. The phase I metabolite 4-OH-AME was found in a sixfold higher concentration in KYSE510 cells than 4-OH-AOH; however, both in a much lower concentration when compared to the parent compounds. Of note, the amount of ROS was increased fourfold by 50 µM 4-OH-AOH as compared to AOH. This is in line with the assumption that catechols might lead to a higher generation of ROS due to redox cycling. Consequently, it would be reasonable to expect 4-OH-AME to produce higher amounts of ROS than AME. However, in the DCF assay, no increase in the fluorescence signal was detected (Fig. 3).
The results of the DCF assay were supported by the data of the Nrf2 reporter gene assay (Fig. 4). Enhanced intracellular levels of ROS are assumed to activate the redox-sensitive transcription factor Nrf2 and subsequent reporter gene expression. Luciferase activity was indeed significantly increased by AOH, AME and 4-OH-AOH, whereas 4-OH-AME turned out to be ineffective. The increase in luciferase activity in the CHO reporter gene system observed for AOH, its hydroxylated metabolite and AME did not completely reflect the DCF data obtained with KYSE510 cells, whereby the different cell types and assay protocols have to be taken into account. The observed Nrf2/ARE-activating potency of AOH in the CHO reporter gene assay is in line with earlier reports on the induction of the Nrf2-pathway in HT29 colon carcinoma cells resulting in enhanced levels of Nrf2/ARE-regulated detoxifying enzymes such as GST and γ-GCL (Tiessen et al. 2013a).
Previously reported by Fehr et al. (2010), the human repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) is a vital factor for the modulation of AOH-mediated DNA damage by reducing covalent topoisomerase–DNA adducts. The study underlines the importance of TDP1 on the repair of topoisomerase-mediated DNA damage which might reduce the stabilized cleavable complexes by AME, 4-OH-AOH and 4-OH-AME. However, another DNA repair process to connect DNA double strands after breakage is non-homologous end joining (NHEJ). The involvement of NHEJ after prolonged incubation in HCT116 cells was examined by Tiessen et al. (2013b). HCT116 cells treated with siRNA to suppress PCNA and Ku70, two proteins involved in the repair process of DNA double strand breaks via NHEJ, were used to determine AOH-mediated DNA lesions. The suppression of Ku70 and PCNA enhanced the DNA-damaging effect of AOH in the comet assay suggesting that this process must be involved in DNA repair. In summary, the lower efficiency of AME, 4-OH-AME and 4-OH-AOH to stabilize topoisomerase II–DNA complexes in association with potential DNA repair processes (NHEJ) and enzymes like TDP1 are feasible explanations for the low or rather non-DNA damage induced by these compounds in KYSE510 cells.
Finally, the role of toxicokinetics and metabolism of these substances in KYSE510 cells was considered. As cytochrome P450 enzymes should occur commonly in esophageal cells (Lechevrel et al. 1999) and Pahlke et al. (2015) reported after 24-h incubation an induction of CYP1A1 transcripts and activity in KYSE510 cells by AOH and (less pronounced) by AME, hydroxylated products of AOH and AME should occur within the cells. However, hydroxylated compounds were neither present in the cell lysate nor in the incubation medium after 1-h exposure, demonstrating that at least the mediated DNA damage by AOH after 1 h can be ascribed to the parent compound. Additionally, glucuronidation and sulfation products of AOH and AME were not detected (Fig. 8) likely due to the short incubation time and low levels of UGTs in esophageal cells (Ohno and Nakajin 2009). After incubation of KYSE510 cells with 4-OH-AOH or 4-OH-AME, the compounds were found in the cell lysate without evidence on the regeneration to AOH or AME in the cell culture medium and cell lysate. The low concentration of 4-OH-AOH and 4-OH-AME in the cell lysate after incubation might be indicative for a poor cellular uptake resulting in the minor effects observed in the ICE- and comet assay. However, only low amounts of the initial concentration (10 µM) of 4-OH-AOH and 4-OH-AME were present in the medium after 1 h, which could be the outcome of further metabolism like glucuronidation, sulfation, GSH-conjugation or methylation. Following this consideration, incubations with 4-OH-AOH exhibited increased amounts of sulfation conjugates in the incubation medium (Fig. 8b) and an additional metabolism product (Supplement Fig. S2). Based on its m/z ratio of 287 and the retention behavior, we speculate that this peak might correspond to a methylated product of 4-OH-AOH generated by catechol-O-methyl transferase (COMT). Indicative for the formation of methylated products is the fact that (1) Burkhardt et al. (2011) described the formation of 4-OH-AOH and 4-OH-AME methylation products at the catechol structure after incubation with rat liver cytosol containing COMT, and (2) COMT has been detected in all human tissues with the highest activity in the liver, followed by kidneys and gastrointestinal tract (Männistö and Kaakkola 1999). Therefore, it seems likely that methylation of 4-OH-AOH is a way of detoxification in KYSE510 cells, which might explain the minor effects in the other assays as compared to AOH. However, it could not be fully excluded that the detected compound belongs to another hydroxyl isomer of AME such as 2-OH-AME. Thus, further studies are needed to confirm the structure of the newly detected metabolite.
In summary, the potential phase I metabolites 4-OH-AOH and 4-OH-AME were synthesized and systematically compared to the parent compounds with respect to oxidative stress, DNA damage, topoisomerase inhibition, cellular uptake and metabolism. The results reveal that in KYSE510 cells the catecholic structure of 4-OH-AOH and 4-OH-AME does not lead to enhanced levels of oxidative DNA damage although the redox cycling activity of 4-OH-AOH is at hand. Despite the huge induction of ROS by 4-OH-AOH, the transcriptional activity of Nrf2 was not affected accordingly. The effects of the metabolites were minor compared to the respective parent substances AOH or AME in terms of topoisomerase inhibition and DNA strand-breaking effects.
Open access funding provided by University of Vienna. This research was financed by the University of Vienna. Mass spectrometric measurements were performed at the Mass Spectrometry Center of the Faculty of Chemistry, University of Vienna.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Austin C, Marsh K (1998) Eukaryotic DNA topoisomerase IIβ. BioEssays 20:215–226. doi:10.1002/(sici)1521-1878(199803)20:3<215:aid-bies5>3.0.co;2-q CrossRefPubMedGoogle Scholar
- Barkai-Golan R (2008) Chapter 8—Alternaria Mycotoxins. In: Mycotoxins in fruits and vegetables. Academic Press, San Diego, pp 185–203. doi:10.1016/B978-0-12-374126-4.00008-5
- Hatahet Z, Kow YW, Purmal AA, Cunningham RP, Wallace SS (1994) New substrates for old enzymes. 5-Hydroxy-2′-deoxycytidine and 5-hydroxy-2′-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2′-deoxyuridine is a substrate for uracil DNA N-glycosylase. J Biol Chem 269:18814–18820PubMedGoogle Scholar
- Liu GT et al (1991) Relationships between Alternaria alternata and oesophageal cancer. IARC Sci Publ 105:258–262Google Scholar
- Liu GT, Qian YZ, Zhang P, Dong WH, Qi YM, Guo HT (1992) Etiologic role of Alternaria alternata in human esophageal cancer. Chin Med J-Peking 105:394–400Google Scholar
- Miao W, Hu L, Scrivens PJ, Batist G (2005) Transcriptional regulation of NF-E2 p45-related factor (NRF2) expression by the aryl hydrocarbon receptor-xenobiotic response element signaling pathway: direct cross-talk between phase I and II drug-metabolizing enzymes. J Biol Chem 280:20340–20348. doi:10.1074/jbc.M412081200 CrossRefPubMedGoogle Scholar
- Pahlke G, Tiessen C, Domnanich K, Kahle N, Groh IA, Schreck I, Weiss C, Marko D (2015) Impact of Alternaria toxins on CYP1A1 expression in different human tumor cells and relevance for genotoxicity. Toxicol Lett 13:30067–30069Google Scholar
- Pelka J, Gehrke H, Esselen M, Türk M, Crone M, Bräse S, Muller T, Blank H, Send W, Zibat V, Brenner P, Schneider R, Gerthsen D, Marko D (2009) Cellular uptake of platinum nanoparticles in human colon carcinoma cells and their impact on cellular redox systems and DNA integrity. Chem Res Toxicol 22:649–659. doi:10.1021/tx800354g CrossRefPubMedGoogle Scholar
- Pfeiffer E, Schmit C, Burkhardt B, Altemöller M, Podlech J, Metzler M (2009) Glucuronidation of the mycotoxins alternariol and alternariol-9-methyl ether in vitro: chemical structures of glucuronides and activities of human UDP-glucuronosyltransferase isoforms. Mycotoxin Res 25:3–10. doi:10.1007/s12550-008-0001-z CrossRefPubMedGoogle Scholar
- Schreck I, Deigendesch U, Burkhardt B, Marko D, Weiss C (2012) The Alternaria mycotoxins alternariol and alternariol methyl ether induce cytochrome P450 1A1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor. Arch Toxicol 86:625–632. doi:10.1007/s00204-011-0781-3 CrossRefPubMedGoogle Scholar
- Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221. doi:10.1002/(sici)1098-2280(2000)35:3<206:aid-em8>3.0.co;2-j CrossRefPubMedGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.