Epoxide Reduction to an Alcohol: A Novel ... - ACS Publications

Jan 15, 2014 - laboratory have disclosed that the epoxide moieties of ATX II and ... present study, this pathway was demonstrated for ATX II in three ...
0 downloads 0 Views 612KB Size
Download PDF
Article pubs.acs.org/crt

Epoxide Reduction to an Alcohol: A Novel Metabolic Pathway for Perylene Quinone-Type Alternaria Mycotoxins in Mammalian Cells Stefanie C. Fleck,† Erika Pfeiffer,† Joachim Podlech,‡ and Manfred Metzler*,† †

Institute of Applied Biosciences, Unit of Food Toxicology, Karlsruhe Institute of Technology (KIT), Karlsruhe D-76131, Germany Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Karlsruhe D-76131, Germany

‡

ABSTRACT: 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.

■

INTRODUCTION Fungi of the genus Alternaria frequently infest food and feed in regions with temperate climate.1,2 An increased incidence of esophageal cancer has been associated with exposure to Alternaria-contaminated grain in regions of China and South Africa,3−5 but which of the more than 70 compounds identified as secondary metabolites of Alternaria fungi are the causative agents remains unknown.1 The toxins generated by Alternaria fungi are commonly classified according to their chemical structures as dibenzo-αpyrones, perylene quinones, fumonisin-like AAL-toxins, and cyclic peptides. Studies on the occurrence, toxicology, and toxicokinetics have so far focused on the dibenzo-α-pyrones alternariol (AOH) and alternariol-9-O-methyl ether (AME).1 Our laboratories have recently become interested in Alternaria toxins with a perylene quinone structure such as altertoxin (ATX) I and II, alteichin (ALTCH), and stemphyltoxin (STTX) III (Scheme 1) because earlier studies have demonstrated that several congeners of this group are highly mutagenic for Salmonella typhimurium even in the absence of a metabolic activation system.6,7 We have reported that ATX II is a very potent mutagen and DNA strand-breaking agent in Chinese hamster V79 cells, a mammalian cell line devoid of most xenobiotic-metabolizing enzyme activities including cytochrome P450.8 Recently, four Alternaria toxins with a perylene quinone structure, ATX I and II, ALTCH, and STTX III, have been studied in the Caco-2 transwell system, which is a widely accepted in vitro model for human intestinal absorption and metabolism.9 Caco-2 cells are derived from a human colonic tumor and, after differentiation, form a monolayer with tight junctions similar to the human intestinal epithelium.10 We © 2014 American Chemical Society

Scheme 1. Structures of the Perylene Quinones ATX I, ATX II, ALTCH, and STTX III

observed that two of the perylene quinones, namely, ATX II and STTX III, which carry an epoxide group (Scheme 1), are Received: October 3, 2013 Published: January 15, 2014 247

dx.doi.org/10.1021/tx400366w | Chem. Res. Toxicol. 2014, 27, 247−253

Chemical Research in Toxicology

Article

three compounds was carried out by using a calibration curve established for the absorbance of ATX II at 254 nm. LC−DAD−MS Analysis. LC−DAD−MS analyses were conducted on a LXQ linear ion trap MSn system (ThermoFisher Scientific, Waltham, MA, USA) using ESI in the negative mode as described earlier.12 A 250 mm × 4.6 mm (i.d.), 5 μm, reversed-phase Luna C8 column (Phenomenex, Torrance, CA, USA) was used with the following gradient (ACN with 0.1% formic acid, deionized water with 0.1% formic acid): 0 min, 40% ACN; 2 min, 40% ACN; 7 min, 50% ACN; 12 min, 70% ACN; 24 min, 70% ACN; 29 min, 100% ACN; 31 min, 100% ACN; 32 min, 40% ACN; and 34 min 40% ACN. The flow rate was 0.5 mL/min, and the detection wavelength was 254 nm. The DAD was calibrated with various dilutions of the stock solution of ATX II. Measurement of DNA Strand Breaks. DNA strand breaks were determined using the alkaline unwinding method as described by Hartwig et al.13 Briefly, 1.7 × 105 cells were seeded in tissue culture plates (40 mm, Biochrom, Berlin) and grown for 24 h (V79 and Caco2) or 48 h (HepG2). Following treatment with the test compounds for 1.5 h, the medium was removed, and the cells were washed with cold phosphate-buffered saline (PBS) and placed on ice. The cells were then treated with an alkaline buffer (pH 12.3) containing 0.03 M NaOH, 0.01 M Na2HPO4, and 0.9 M NaCl for 30 min in the dark at 20 °C to allow the DNA to unwind. Subsequently, the solution was neutralized to pH 6.8 with 0.1 M HCl, sonicated for 15 s on ice, and treated with sodium dodecyl sulfate (final concentration, 0.06%). Separation of single- and double-stranded DNA was carried out on hydroxylapatite columns at 60 °C. The DNA was stained with Hoechst 33258 dye (final concentration, 7.7 × 10−7 M), and the resulting fluorescence was measured at 465 nm (excitation wavelength, 360 nm). Depletion of GSH Content. The intracellular GSH level was measured according to the method of Tietze.14 The depletion of GSH content was achieved by preincubating 1.7 × 105 V79 cells for 24 h with a 100 μM solution of buthionine sulfoximine (BSO) in DMEM containing 1% DMSO. The depleted cells were then incubated with the test compounds and analyzed for DNA strand breaks as described above. For the measurement of the GSH level in normal and depleted cells, two tissue culture plates with a diameter of 40 mm were combined to obtain an adequate number of cells. GSH content is calculated as nanomoles of GSH per 106 cells.

metabolized in Caco-2 cells, whereas ATX I and ALTCH are not.9 The metabolite of ATX II could be unequivocally identified as ATX I using LC−DAD−MS and an authentic reference compound, indicating that the epoxide ring has been opened and the oxygen has been reduced to a hydroxyl group. Likewise, STTX III was metabolized in Caco-2 cells via epoxide reduction to the alcohol ALTCH. In the present study, we have investigated whether this novel pathway in the metabolism of xenobiotics also occurs in other mammalian cells, and we attempted to elucidate the molecular mechanism of the reduction of the epoxide to an alcohol. Moreover, ATX II was clearly shown to react chemically with glutathione and other thiol-containing molecules, and the structures of the reaction products were tentatively identified.

■

EXPERIMENTAL PROCEDURES

Chemicals. ATX I and ATX II were isolated from a culture of Alternaria alternata as described in detail by Fleck et al.8 Briefly, A. alternata was grown for 24 days on a medium containing rice flour, and the homogenized mycelium was extracted with ethyl acetate. The toxins were then purified by preparative high-performance liquid chromatography (HPLC) on a reversed-phase C18 column. Identification was achieved by comparison with data published in the literature using mass spectrometry (MS) with electrospray ionization (ESI) and tandem MS as well as UV and NMR spectroscopy.8,9 Both compounds had a purity of >98% according to analysis using HPLC with diode array detection (DAD). ATX I and ATX II were quantified by measuring their UV absorbance in methanol at the following wavelengths using the molar extinction coefficients published by Stack et al:11 ATX I, 256 (3.46 × 104 M−1 cm−1) and 285 nm (1.63 × 104 M−1 cm−1) and ATX II, 258 (3.17 × 104 M−1 cm−1) and 358 nm (5.3 × 103 M−1 cm−1). Stock solutions of both toxins were prepared in ethanol. Other chemicals and reagents were obtained from Sigma-Aldrich/ Fluka (Taufkirchen, Germany) and were of the highest quality available. HPLC-grade acetonitrile (ACN) and methanol were from Roth (Karlsruhe, Germany). Cell Lines and Incubations with ATX II. Caco-2, HCT 116, HepG2, and V79 cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). For better comparability, all cells were cultured in Dulbecco’s modified Eagle’s medium containing Ham’s F-12 nutrient mixture (DMEM/ F12) supplemented with 10% fetal calf serum (FCS, Invitrogen, Karlsruhe, Germany), 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Cells were cultured at 37 °C in a water-saturated atmosphere containing 5% carbon dioxide. For metabolic studies, 2.5 × 105 cells were incubated with 1 μM (2 nmol) ATX II for 1.5 h in serum-free DMEM/F12 followed by extraction of the medium with ethyl acetate and analysis of the extract using LC−DAD−MS as described in detail by Fleck et al.9 To account for losses resulting from extraction and decomposition of ATX II, two control incubations were carried out in the absence of cells, and the toxin was extracted either immediately (control 1) or after 1.5 h (control 2). Chemical Reaction of ATX II with Monothiol and Dithiol Compounds. Phosphate buffer (0.2 mL, 0.1 M, pH 7.4) containing 10 μM (2 nmol) ATX II and 1 mM mercaptoethanol (ME), Nacetylcysteine (NAC), or glutathione (GSH) was kept at 37 °C for 30 min. The products of the reaction were subsequently extracted with 0.5 mL of ethyl acetate after adding 0.2 mL of 0.7 M glycine-HCl buffer, pH 1.2. The incubations of ATX II with the dithiol compounds ethane-1,2dithiol, propane-1,3-dithiol, dihydrolipoic acid, butane-1,4-dithiol, dithiothreitol, and pentane-1,5-dithiol were carried out under the same conditions as described for the monothiol compounds ME, NAC, and GSH. After addition of glycine-HCl buffer and extraction with 0.5 mL of ethyl acetate, the amounts of ATX II, ATX I, and monoadducts were measured by LC−DAD−MS. Quantitation of all

■

RESULTS Epoxide Reduction of ATX II in Various Mammalian Cell Types. The observation that ATX II is metabolized to ATX I in cultured Caco-2 cells9 raised the question as to whether the same pathway is active in other mammalian cell lines. Therefore, ATX II was incubated with human colon cells HCT 116, human liver cells HepG2, and Chinese hamster V79 lung fibroblasts. Analysis of the incubation medium by LC− DAD−MS analysis showed that all four cell types were able to generate ATX I from ATX II (Table 1). A typical HPLC profile of the extract of the incubation with HepG2 cells is depicted in Figure 1 (bottom) together with the profile of the corresponding control incubation of ATX II in the absence of cells (top). The peak designated as ATX I was identified by its UV, ESI-MS, and MS/MS spectrum as described by Fleck et al.9 It also cochromatographed with an authentic ATX I standard. No ATX I could be detected in the control incubations of ATX II without cells because the small peak at 12.1 min had the retention time of ATX I but did not contain the ions of ATX I in ESI-MS. The mass spectra of the other peaks present in the chromatograms of the complete and the control incubation did not suggest perylene quinone structures. From such control incubations, 85.7% of the applied ATX II could be recovered immediately after addition to the medium (control 1) and 71.5%, after 1.5 h (control 2), suggesting that 248

dx.doi.org/10.1021/tx400366w | Chem. Res. Toxicol. 2014, 27, 247−253

Chemical Research in Toxicology

Article

Table 1. Epoxide Reduction of ATX II to ATX I in Various Cell Linesa cell line no cells (control 1)c no cells (control 2)e Caco-2 HCT 116 HepG2 V79

ATX II (nmol)

ATX I (nmol)

total recovery (%)b

± ± ± ± ± ±

not detectedd not detectedd 1.10 ± 0.15 0.39 ± 0.02 0.49 ± 0.03 0.06 ± 0.02

86 71 (set as 100%) 84f 73f 83f 45f

1.72 1.43 0.21 0.62 0.80 0.64

0.06 0.37 0.04 0.11 0.11 0.19

a

ATX II (2 nmol) was incubated and analyzed as described in the legend of Figure 1. The recovery in cell-free control incubations for 1.5 h (control 2) was set as 100% for the incubations with cells. Data represent the mean ± standard deviation of at least three independent experiments. bSum of ATX I and ATX II. cExtracted before incubation. dLess than 0.001 nmol. eExtracted after incubation. f Percent of 1.43 nmol (control 2).

Figure 2. HPLC profile of the extract of the incubation of ATX II with ME.

The [M − H]− ions of each of these products correspond to a monothiol adduct of ATX II, which had eliminated two molecules of water (i.e., m/z 391 for the ME adduct, m/z 476 for the NAC adduct, and m/z 620 for the GSH adduct). The fragment ions in the MS2 of the [M − H]− ions indicate that the adducts contain the respective thiol compounds (Figure 3). The exact chemical structure of these adducts (i.e., the positions of the two additional double bonds and the sites of the sulfur) are not yet known. The elimination of two water molecules from the primary adduct could either lead to phenanthrene or to perylene derivatives. However, the formation of completely aromatic perylene structures is considered unlikely because of the UV−vis absorption of the ME, NAC, and GSH adducts, which are very similar. As an example, the UV spectrum of the ME adduct is depicted in Figure 4B. This adduct exhibits a yellow color with an absorption maximum at 440 nm. Unsubstituted perylene has a reported absorption maximum at 436 nm,15 and additional hydroxyl groups and the thioether group would shift this maximum to much longer wavelengths. For example, a thioether group causes a shift of 30 nm according to the empirical rules by Woodward and Fieser.15 Because the absorption maxima of the ATX II adducts with ME, NAC, and GSH are at wavelengths too low for perylene derivatives, we propose the structures of substituted phenanthrenes. A possible mechanism of formation is depicted in Scheme 2. However, the adduct structures must be considered tentative and need confirmation (e.g., by NMR spectroscopy). In contrast to the facile elimination of two molecules of water from the primary monothiol adducts of ATX II (Scheme 2), the structurally similar ATX I is a stable molecule. To rule out that the water elimination depicted in Scheme 2 was caused by the low pH during extraction (see Experimental Procedures), the extraction was also carried out at neutral pH. The same dehydrated product and no primary adduct were obtained. Moreover, the dehydrated product is formed prior to extraction as shown by the UV−vis spectrum of the incubation mixture, and ATX I was also stable under the conditions of acidic extraction (data not shown). Therefore, we propose that the instability of the primary adduct with ATX II is caused by the sulfanyl substituent, which is lacking in ATX I. Reaction of ATX II with Dithiol Compounds. As described earlier, ATX II undergoes an epoxide reduction to ATX I in all mammalian cells studied so far (Table 1). This is not a common metabolic reaction for epoxides, and its molecular mechanism is not known. However, a clue for a

Figure 1. Metabolism of ATX II in cultured HepG2 cells. One micromolar ATX II (total amount, 2 nmol) was incubated in serumfree DMEM/F12 either in the absence (top) or presence (bottom) of 2.5 × 105 cells for 1.5 h at 37 °C. The incubation media was then extracted with ethyl acetate, and the extracts were analyzed with LC− DAD−MS.

ATX II is not completely stable under the incubation conditions. All of the incubations conducted in the presence of cells contained ATX I, although at various amounts (Table 1). The highest yield of ATX I was obtained with Caco-2 cells followed by HepG2, HCT116, and V79 cells. The sum of the amounts of ATX I and ATX II recovered from Caco-2 and HepG2 cells almost accounted for the recovery of ATX II from the respective control incubations (control 2), whereas more than half of the amount of ATX II could not be accounted for in V79 cells (Table 1), which may be due to unknown metabolites or reaction with cellular macromolecules. Reaction of ATX II with Thiol Groups. To probe its reactivity with thiol compounds, ATX II was incubated with each of the three monothiol compounds: 2-mercaptoethanol (ME), N-acetylcysteine (NAC), and glutathione (GSH). LC− DAD−MS analysis of the extract of the incubation mixture showed that a major product had formed with each of the monothiols, as exemplified for the ME adduct in Figure 2. 249

dx.doi.org/10.1021/tx400366w | Chem. Res. Toxicol. 2014, 27, 247−253

Chemical Research in Toxicology

Article

was incubated with several dithiol compounds differing in the distance of the two thiol groups and carrying substituents. The results are summarized in Figure 5. Whereas ethane-1,2-dithiol gave rise to minute amounts of a monothiol adduct but no ATX I, a large amount of ATX I but no monothiol adduct was found with propane-1,3-dithiol. The same result was obtained with dihydrolipoic acid, a natural substituted propane-1,3-dithiol. Butane-1,4-dithiol and pentane-1,5-dithiol, in which the distance between the two thiol group increases, preferred the formation of a monothiol adduct with very little and no formation of ATX I, respectively. However, the substituted butane-1,4-dithiol DTT gave rise to a high amount of ATX I without a detectable monothiol adduct (Figure 5). Consequences of Epoxide Reduction and GSH Conjugation for the Genotoxicity of ATX II. Because ATX II is a direct-acting genotoxin not requiring metabolic activation for its mutagenic and DNA strand-breaking effects,6,8 any metabolic alteration of the chemical structure must be expected to affect genotoxicity. To assess the effect of epoxide reduction, the DNA strand-breaking activities of ATX II and ATX I were compared in V79, HepG2, and Caco-2 cells. AOH was included for comparison. In studies using various concentrations of the three toxins, the DNA strand-breaking activities of ATX II, ATX I, and AOH were found to be concentration-dependent, as reported for V79 cells by Fleck et al.8 At concentrations high enough to evoke a clear DNA strand-breaking effect but still in the dynamic range of the concentration-effect curve, the following order of activity was observed: ATX II ≫ ATX I ≈ AOH (Figure 6). Thus, the conversion of ATX II to ATX I does not represent a complete metabolic detoxification but merely an attenuation of genotoxicity, as ATX I is still a decent DNA strand-breaking agent with about the same activity as AOH. Although the three cell lines exhibit different rates of metabolic conversion of ATX II to ATX I (Table 1), no statistically significant differences were observed for the extent of DNA strand breaks induced by ATX II (Figure 6). The effect of adduct formation with GSH on the DNA strand-breaking activity of ATX II was assessed in V79 cells by comparing cells with and without depletion of GSH. Treatment with 100 μM buthionine sulfoximine (BSO) for 24 h is known to abolish virtually all cellular GSH by inhibiting γglutamylcysteine synthetase, the enzyme involved in the first step of GSH biosynthesis16 (Figure 7, left). When the DNA strand breaks induced by ATX II in normal and BSO-treated cell were compared, only a marginal and nonstatistically significant increase was observed in GSH-depleted cells (Figure

Figure 3. MS2 of the [M − H]− ions of the adducts of ATX II with ME (A), NAC (B), and GSH (C).

putative mechanism was obtained when ATX II was incubated with the dithiol compound dithiothreitol (DTT) in the course of studies on the reaction of ATX II with thiols. Only trace amounts of the expected monoadduct of ATX II with DTT were obtained, but large amounts of ATX I were obtained. In a systematic investigation of this surprising observation, ATX II

Figure 4. Measured UV spectra of ATX II (A) and its monothiol adduct with mercaptoethanol (B). 250

dx.doi.org/10.1021/tx400366w | Chem. Res. Toxicol. 2014, 27, 247−253

Chemical Research in Toxicology

Article

Scheme 2. Mechanism Proposed for the Reaction of ATX II with Monothiols

moiety to a monohydroxyl group, resulting in the formation of ATX I. The molecular mechanism underlying this epoxide reduction is presently not known. The same reaction (i.e., conversion of ATX II to ATX I) was found to take place when ATX II was chemically reacted with certain dithiol compounds. On the basis of the observation that only 1,3- and 1,4-dithiols lead to the formation of ATX I (Figure 5), we propose the mechanism depicted in Scheme 3. Following the initial formation of a thiol adduct analogous to those observed with monothiols, adducts from 1,3-dithiols can form a fivemembered ring and use the second thiol group for an intramolecular reduction reaction, yielding ATX I and the oxidized form of the dithiol. This intramolecular reduction is less favorable for adducts with 1,4-dithiols and does not occur at all with adducts of 1,2-dithiols or 1,5-dithiols. The finding that no significant amounts of a monoadduct of ATX II could be observed in the reaction with ethane-1,2-dithiol may be due to the further reaction of an intermediate monoadduct with another ATX II molecule to yield an adduct with two crosslinked ATX II molecules, which would not have been detected by our LC−DAD−MS analysis. The observation that the substituted 1,4-dithiol DTT is more suited for the intramolecular reaction than the unsubstituted butane-1,4-dithiol (Figure 5) may be explained by the fact that the ring closure is generally favored in substituted compounds because a decrease of the carbon chain’s interior angle facilitates a cyclization reaction.17 A similar mechanism as that shown in Scheme 3 for the reaction of ATX II with suitable dithiols has been proposed for the chemical reaction of vitamin K epoxide with DTT.18,19 Vitamin K is a substituted 1,4-naphthoquinone. Its olefinic epoxide first reacts with one of the thiol groups of DTT to form a monoadduct. Subsequently, the DTT monoadduct undergoes an intramolecular reduction, resulting in the elimination of oxidized DTT, and formation of an enolate intermediate, which upon protonation gives rise to the hydroxylated form of vitamin K. This reaction sequence is completely analogous to the mechanism proposed for ATX II in Scheme 3. In the case of vitamin K, the primary monoadduct with DTT and the hydroxyl compound have been unambiguously identified by NMR spectra.19 A similar molecular mechanism is assumed for the enzymatic reduction of vitamin K epoxide: The enzyme vitamin K epoxide reductase uses a reduced disulfide in its active site instead of DTT and converts the resulting hydroxy compound to vitamin K through the elimination of water.18−20 In the studies on the chemical conversion of vitamin K epoxide to the hydroxyl compound, the formation of two regioisomers of the latter was observed because of the attack of the thiol at each of the two C-atoms of the epoxide group.19 Our chemical and cell culture studies with ATX II gave rise to

Figure 5. Reaction products of ATX II with various dithiol compounds. Two nanomoles (1 μM) of ATX II in 0.1 M phosphate buffer, pH 7.4, were incubated for 30 min at 37 °C with 2 μmol (1 mM) of the dithiol compound indicated. The incubation mixture was then extracted with ethyl acetate, and total amounts of ATX II, ATX I, and the monoadduct were determined by LC−DAD−MS/MS. Data represent the mean ± standard deviation of at least three independent experiments. c, control (without dithiol).

Figure 6. DNA strand-breaking activity of ATX II, ATX I, and AOH in V79, HepG2, and Caco-2 cells. The cells were treated with the toxins at the indicated concentrations for 1.5 h, and the DNA strand breaks were determined using the alkaline unwinding assay. Data represent the mean ± standard deviation of at least three independent experiments.

7, right). The DNA strand-breaking activity of AOH, which does not form GSH adducts, was not affected by depletion of GSH, in contrast to 1,4-naphthoquinone, which was used as a positive control.

■

DISCUSSION This study has addressed the hitherto unknown in vitro metabolism of ATX II, a perylene quinone-type Alternaria mycotoxin with an epoxide group. In four mammalian cell lines, the only metabolic reaction detectable by LC−DAD−MS analysis under our conditions was the conversion of the epoxide 251

dx.doi.org/10.1021/tx400366w | Chem. Res. Toxicol. 2014, 27, 247−253

Chemical Research in Toxicology

Article

Figure 7. DNA strand-breaking activity of ATX II and AOH in V79 cells without and with depletion of GSH. 1,4-Naphthoquinone was used as a positive control. Left: Effect of treatment with increasing concentrations of BSO for 24 h on the GSH level. Right: DNA strand breaks in nondepleted cells (blank columns) and in GSH-depleted cells (filled columns) after treatment with the test compounds for 1.5 h. Data represent the mean ± standard deviation of at least three independent experiments.

Scheme 3. Mechanism Proposed for the Epoxide Reduction of ATX II by Dithiols

only one peak in LC−DAD−MS analysis, which cochromatographed with authentic ATX I (Figure 1). The attack of the thiol at position C-7a of ATX II is more likely than at C-8a because of less sterical hindrance at C-7a (Scheme 3). However, because no reference compound for the regioisomer of ATX I exists, it cannot be ruled out that the peak considered as ATX I also contains the isomeric alcohol, which would probably have the same ESI mass spectrum as ATX I. Further studies are needed to clarify the regio- and stereospecificity of the epoxide reduction of ATX II. The enzyme system involved in the epoxide reduction of ATX II to ATX I in mammalian cells is presently unknown. Preliminary studies in our laboratory have shown that this metabolic reaction is not catalyzed by microsomes or cytosol from rat liver but by the mitochondrial fraction from piglet liver (unpublished data), which may suggest the involvement of dihydrolipoic acid-containing mitochondrial enzymes. This would also be of interest with respect to the high cytotoxicity of ATX II,9 which may be explained if mitochondrial enzymes involved in cellular respiration are affected (e.g., pyruvate dehydrogenase and α-ketoglutarate dehydrogenase). The reaction of ATX II has also been studied with compounds containing only one thiol group, such as GSH. The resulting initial thiol adduct (Scheme 2) is unstable and spontaneously eliminates two molecules of water. The proposed structure, which was derived from UV and mass spectra, remains tentative until it is confirmed by NMR spectra. In particular, the position of the sulfanyl substituent is not certain because an initial attack of the thiol group at C-8a of ATX II cannot be excluded.

It is of interest to note that the genotoxicity of ATX II was not significantly different between cells with a different metabolic activity for ATX I formation (Figure 6) or in cells after depletion of GSH (Figure 7), although ATX I is less genotoxic by a factor of about 20 than ATX II and the GSH adduct of ATX II is presumably nongenotoxic. A possible explanation for the failure of the cells to inactivate ATX II under our experimental conditions may be that both the epoxide reduction and the conjugation with GSH are slow in comparison with the induction of genetic damage, but this speculation needs to be substantiated in further studies. At present, epoxide reduction and formation of a GSH conjugate are the only known metabolic pathways for ATX II. In preliminary studies in our laboratory, ATX II was incubated with rat hepatic microsomes fortified with the cofactors for monooxygenation and glucuronidation, and a LC−MS search for the products of hydroxylation and epoxide hydrolysis as well as for glucuronides of ATX II was conducted by extracting the corresponding [M − H]− ions from the total ion chromatograms. Under conditions where analogous studies21−23 with AOH and AME had shown extensive hydroxylation and glucuronidation, no such metabolites could be detected with ATX II (unpublished data). These preliminary observations, which suggest that hydroxylation, glucuronidation, and epoxide hydrolysis may be minor pathways or even absent in the metabolism of ATX II, need to be confirmed and extended in further studies. ATX II is a highly potent and direct-acting mutagen, as demonstrated in previous investigations.6,8 Together with the possible lack of efficient metabolic inactivation, ATX II may be 252

dx.doi.org/10.1021/tx400366w | Chem. Res. Toxicol. 2014, 27, 247−253

Chemical Research in Toxicology

Article

(11) Stack, M. E., Mazzola, E. P., Page, S. W., Pohland, A. E., Highet, R. J., Tempesta, M. S., and Corley, D. G. (1986) Mutagenic perylenequinone metabolites of Alternaria alternata: Altertoxins I, II, and III. J. Nat. Prod. 49, 866−871. (12) Pfeiffer, E., Hildebrand, A., Damm, G., Rapp, A., Cramer, B., Humpf, H. U., and Metzler, M. (2009) Aromatic hydroxylation is a major metabolic pathway of the mycotoxin zearalenone in vitro. Mol. Nutr. Food. Res. 53, 1123−1133. (13) Hartwig, A., Dally, H., and Schlepegrell, R. (1996) Sensitive analysis of oxidative DNA damage in mammalian cells: Use of the bacterial Fpg protein in combination with alkaline unwinding. Toxicol. Lett. 88, 85−90. (14) Tietze, F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem. 27, 502−522. (15) Hesse, M., Meier, H., and Zeeh, B. (1997) Spectroscopic Methods in Organic Chemistry, Georg Thieme Verlag, Stuttgart, Germany. (16) Griffith, O. W., and Meister, A. (1979) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine(S-n-butyl homocysteine sulfoximine). J. Biol. Chem. 254, 7558−7560. (17) Beesley, R. M., Ingold, C. K., and Thorpe, J. F. (1915) CXIX.− The formation and stability of spiro-compounds. Part I. Spirocompounds from cyclohexane. J. Chem. Soc. 107, 1080−1106. (18) Silverman, R. B. (1981) Chemical model studies for the mechanism of vitamin K epoxide reductase. J. Am. Chem. Soc. 103, 5939−5941. (19) Preusch, P. C., and Suttie, J. W. (1983) A chemical model for the mechanism of vitamin K epoxide reductase. J. Org. Chem. 48, 3301−3305. (20) Van Horn, W. D. (2013) Structural and functional insights into human vitamin K epoxide reductase and vitamin K epoxide reductaselike1. Crit. Rev. Biochem. Mol. Biol. 48, 357−372. (21) Pfeiffer, E., Schebb, N. H., Podlech, J., and Metzler, M. (2007) Novel oxidative in vitro metabolites of the mycotoxins alternariol and alternariol methyl ether. Mol. Nutr. Food Res. 51, 307−316. (22) Pfeiffer, E., Burkhardt, B., Altemöller, M., Podlech, J., and Metzler, M. (2008) Activities of human recombinant cytochrome P450 isoforms and human hepatic microsomes for the hydroxylation of Alternaria toxins. Mycotoxin Res. 24, 117−123. (23) Pfeiffer, E., Schmit, C., Burkhardt, B., Altemöller, M., Podlech, J., and 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.

a candidate for the etiological agent of the esophageal cancer purportedly associated with exposure to Alternaria-contaminated food.3−5 Although frequently found in cultures of Alternaria spp. isolated from moldy products, there are regretably no data on the occurrence of ATX II or other perylene quinone-type Alternaria toxins in food and feed items.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +49 721 608 42132. Fax: +49 721 608 47255. E-mail: [email protected]. Funding

This study was funded by the “Food and Health” research program of KIT as well as by a fellowship from the state of Baden-Württemberg to S.C.F. Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS ACN, acetonitrile; ALTCH, alteichin; AME, alternariol-9-Omethyl ether; AOH, alternariol; ATX, altertoxin; DAD, diode array detector; DMEM/F12, Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham; DMSO, dimethyl sulfoxide; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen; ESI, electrospray ionization; FCS, fetal calf serum; HBSS, Hank’s buffered salt solution; HPLC, highperformance liquid chromatography; MS, mass spectra, mass spectrometry; PBS, phosphate-buffered saline; STTX, stemphyltoxin

■

REFERENCES

(1) EFSA Panel on Contaminants in the Food Chain (CONTAM) (2011) Scientific opinion on the risks for animal and public health related to the presence of Alternaria toxins in feed and food. EFSA J. 9, 2407. (2) Ostry, V. (2008) Alternaria mycotoxins: An overview of chemical characterization, producers, toxicity, analysis and occurrence in foodstuffs. World Mycotoxin J. 1, 175−188. (3) Liu, G. T., Qian, Y. Z., Zhang, P., Dong, Z. M., Shi, Z. Y., Zhen, Y. Z., Miao, J., and Xu, Y. M. (1991) Relationships between Alternaria alternata and oesophageal cancer. IARC Sci. Publ. 105, 258−262. (4) Liu, G. T., Qian, Y. Z., Zhang, P., Dong, W. H., Qi, Y. M., and Guo, H. T. (1992) Etiological role of Alternaria alternata in human esophageal cancer. Chin. Med. J. 105, 394−400. (5) Panigrahi, S. (1997) Alternaria toxins, In Handbook of Plant and Fungal Toxicants (D’Mello, J. P. F., Ed.) pp 319−337, CRC Press, Boca Raton, FL. (6) Stack, M. E., and Prival, M. J. (1986) Mutagenicity of the Alternaria metabolites altertoxins I, II, and III. Appl. Environ. Microbiol. 52, 718−722. (7) Davis, V. M., and Stack, M. E. (1991) Mutagenicity of stemphyltoxin III, a metabolite of Alternaria alternata. Appl. Environ. Microbiol. 57, 180−182. (8) Fleck, S. C., Burkhardt, B., Pfeiffer, E., and Metzler, M. (2012) Alternaria toxins: Altertoxin II is a much stronger mutagen and DNA strand breaking mycotoxin than alternariol and its methyl ether in cultured mammalian cells. Toxicol. Lett. 214, 27−32. (9) Fleck, S. C., Pfeiffer, E., and Metzler, M. (2013) Permeation and metabolism of Alternaria mycotoxins with perylene quinone structure in cultured Caco-2 cells. Mycotoxin Res. [Online early acces], DOI: 10.1007/s12550-013-0180-0, Published Online: Oct 31, 2013. (10) Press, B., and Di Grandi, D. (2008) Permeability for intestinal absorption: Caco-2 assay and related issues. Curr. Drug Metab. 9, 893− 900. 253

dx.doi.org/10.1021/tx400366w | Chem. Res. Toxicol. 2014, 27, 247−253