Glucuronidation of the mycotoxins alternariol and alternariol-9-methyl ether in vitro: chemical structures of glucuronides and activities of human UDP-glucuronosyltransferase isoforms

Alternariol (AOH) and alternariol-9-methyl ether (AME) are major toxins produced by fungi of the genus Alternaria and are frequently found in various food items. Because AOH has three hydroxyl groups and AME two, the formation of various glucuronides must be expected. When AOH was incubated with hepatic and intestinal microsomes from rats, pigs and humans in the presence of uridine diphosphate glucuronic acid, two glucuronides were detected and tentatively identified as AOH-3-O-glucuronide and AOH-9-O-glucuronide. Under the same conditions, AME yielded predominantly AME-3-O-glucuronide and only small amounts of AME-7-O-glucuronide. The activities of all microsomes for the glucuronidation of AOH and AME were in the same range. Nine out of ten recombinant human UDP-glucuronosyltransferases (UGTs) were able to glucuronidate AOH, and eight out of ten UGTs had activity for AME. These data suggest that AOH and AME are readily glucuronidated in hepatic and extrahepatic tissues, implying that glucuronidation constitutes a major metabolic pathway in the disposition of these mycotoxins. Presented at the Mycotoxin Workshop, Utrecht, Netherlands, April 28–30, 2008     

Permeation and metabolism of Alternaria mycotoxins with perylene quinone structure in cultured Caco-2 cells

The absorption of four Alternaria toxins with perylene quinone structures, i.e. altertoxin (ATX) I and II, alteichin (ALTCH) and stemphyltoxin (STTX) III, has been determined in the Caco-2 cell Transwell system, which represents a widely accepted in vitro model for human intestinal absorption and metabolism. The cells were incubated with the four mycotoxins on the apical side, and the concentration of the toxins in the incubation media of both chambers and in the cell lysate were determined by liquid chromatography coupled with diode array detection and mass spectrometry (LC-DAD-MS) analysis. ATX I and ALTCH were not metabolised in Caco-2 cells, but ATX II and STTX III were partly biotransformed by reductive de-epoxidation to the metabolites ATX I and ALTCH, respectively. Based on the apparent permeability coefficients (P_app), the following ranking order for the permeation into the basolateral compartment was obtained: ATX I > ALTCH >> ATX II > STTX III. Total recovery of the four toxins decreased in the same order. It is assumed that the losses of STTX III, ATX II and ALTCH in Caco-2 cells are caused by covalent binding to cell components due to the epoxide group and/or the α,β-unsaturated carbonyl group present in these toxins. We conclude from this study that ATX I and ALTCH are well absorbed from the intestinal lumen into the portal blood in vivo. For ATX II and STTX III, intestinal absorption of the parent toxins is very low, but these toxins are partly metabolised to ATX I and ALTCH, respectively, in the intestinal epithelium and absorbed as such.     

Light inhibits the production of alternariol and alternariol monomethyl ether in Alternaria alternata

Alternaria alternata (Fr.) Keissler, grown in drop culture, produced alternariol and alternariol monomethyl ether in late growth phase. Production was almost completely inhibited when the fungal cultures were exposed to white light (180 W/m2), although mycelial dry weight was not significantly affected. The fungus was most sensitive to light during the exponential growth phase. Twelve hours of light exposure was sufficient to decrease significantly the production of the secondary metabolites. In light the fungus produced a red-brown pigment of unknown nature.     

Alternaria toxins alternariol and alternariol monomethyl ether in grain foods in Canada

Alternaria alternata has been reported to be the most common fungus on Canadian Western wheat. The Alternaria toxins alternariol (AOH) and alternariol monomethyl ether (AME) are mutagenic in vitro and there is also limited evidence for carcinogenic properties. They have been found in wheat from Europe, Argentina, China and Australia, but they have not been looked for in Canadian grains or grain foods. In the present study, 83 samples of grain-based food sold in Canada, including flour, bran, breakfast cereals, infant cereals and bread, were analysed for AOH and AME using extraction with methanol, clean-up on combined aminopropyl/C18 solid phase extraction (SPE) columns, and liquid chromatography (LC) with tandem mass spectrometric (MS/MS) determination. The overall average recoveries of AOH and AME from a variety of spiked cereal foods (n?=?13) were 45?±?9?% and 53?±?9?%, which could be attributed mainly to MS matrix effects The instrumental limits of detection (LOD) were 0.34?ng/g and 0.13?ng/g for AOH and AME, respectively, and the instrumental limits of quantitation (LOQ) were 1.1 and 0.43?ng/g. Of 83 samples analysed, 70 were positive for AOH (up to 63?ng/g, in a soft wheat bran) and 64 contained AME (up to 12?ng/g in a bran-based breakfast cereal). Of particular interest was the presence of AOH and/or AME in 27 out of 30 infant foods (up to 4.4?ng/g and 9.0?ng/g, respectively, in a sample of multigrain cereal).     

Natural occurrence of alternariol and alternariol monomethyl ether in soya beans

The natural occurrence of alternariol (AOH) and alternariol monomethyl ether (AME) in soya beans harvested in Argentina was evaluated. Both toxins were simultaneously detected by using HPLC analysis coupled with a solid phase extraction column clean-up. Characteristics of this in-house method such as accuracy, precision and detection and quantification limits were defined by means of recovery test with spiked soya bean samples. Out of 50 soya bean samples, 60% showed contamination with the mycotoxins analyzed; among them, 16% were only contaminated with AOH and 14% just with AME. Fifteen of the positive samples showed co-occurrence of both mycotoxins analyzed. AOH was detected in concentrations ranging from 25 to 211?ng/g, whereas AME was found in concentrations ranging from 62 to 1,153?ng/g. Although a limited number of samples were evaluated, this is the first report on the natural occurrence of Alternaria toxins in soya beans and is relevant from the point of view of animal public health.     

Light inhibits the production of alternariol and alternariol monomethyl ether in Alternaria alternata.

Alternaria alternata (Fr.) Keissler, grown in drop culture, produced alternariol and alternariol monomethyl ether in late growth phase. Production was almost completely inhibited when the fungal cultures were exposed to white light (180 W/m2), although mycelial dry weight was not significantly affected. The fungus was most sensitive to light during the exponential growth phase. Twelve hours of light exposure was sufficient to decrease significantly the production of the secondary metabolites. In light the fungus produced a red-brown pigment of unknown nature.     

Evaluation of alternariol and alternariol methyl ether for mutagenic activity in Salmonella typhimurium.

Alternariol and alternariol methyl ether were tested in the Ames Salmonella typhimurium assay, and both were shown, with and without metabolic activation, to be nonmutagenic to strains TA98 and TA100. The finding of other investigators that alternariol methyl ether is weakly mutagenic to TA98 without metabolic activation could have resulted from the presence of a small amount of one of the highly mutagenic altertoxins in the alternariol methyl ether originally tested.     

Occurrence of alternariol, alternariol monomethyl ether and tenuazonic acid in Argentinean tomato puree

The occurrence ofAlternaria mycotoxins was investigated in 80 samples of tomato puree processed and sold in Argentina. Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) were searched for by liquid chromatography. Thirty-nine of the 80 samples showed mycotoxin contamination. TA was found in 23 samples (39-4021 μg/kg), AOH in 5 samples (187-8756 μg/kg), and AME in 21 samples (84-1734 μg/kg). Co-occurrence of two of these toxins was detected in 10 samples. This is the first report of natural occurrence of AOH, AME and TA in tomato products in Argentina.     

Occurrence of alternariol and alternariol monomethyl ether in beverages from the Entre Rios Province market, Argentina

One hundred and eighty five samples of red, white and rosé wines and different juices purchased in Entre Rios, Argentina, were analyzed for the Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME). White wines were analyzed after removal of alcohol by a nitrogen stream and concentrated. AOH in red wines was cleaned up by solid-phase extraction columns in series (octadecyl and amino propyl modified silica) and AME quantified directly on the sample. The juices were filtered and concentrated, and then all sample extracts were quantified by high performance liquid chromatography with photodiode array detector that allows confirmation through UV spectra. Method validation revealed a good sensitivity with adequate LOD and LOQ for AME and less sensitivity for AOH (i.e. white wine: AME 0.8 and 1.4 ng/mL, AOH 2 and 3.3 ng/mL; red wine: AME 0.1 and 0.2 ng/mL, AOH 4.5 and 7.5 ng/mL; apple juice: AME 1.7 and 2.8 ng/mL, AOH 5 and 9 ng/mL; other juices: AME 2.0 and 3.1 ng/mL, AOH 6 and 10 ng/mL). Recoveries in all cases were greater than 80 %. Four of 53 white wine samples were contaminated with AOH with a maximum level of 18 ng/mL, 6 of 56 samples of red wine had a maximum of 13 ng/mL, and 3 of 68 samples of juices had traces of AOH. AME was less frequently detected than AOH, and the LOD and LOQ for AME are smaller than for AOH. Only three samples of white wine and one of red wine were contaminated, but in only one white wine sample (AME 225 ng/mL) did the toxin level exceed the LOQ.     

Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells

Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that rotavirus infection of Caco-2 cells caused disruption of tight junctions and loss of transepithelial resistance (TER) in the absence of cell death. TER declined from 300 to 22 Omega. cm(2) between 8 and 24 h after infection and was accompanied by increased transepithelial permeability to macromolecules of 478 and 4,000 Da. Distribution of tight junction proteins claudin-1, occludin, and ZO-1 was significantly altered during infection. Claudin-1 redistribution was notably apparent at the onset of the decline in TER. Infection was associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% of control at 24 h after infection), conditions known to reduce the integrity of epithelial tight junctions. In conclusion, these data show that rotavirus infection of Caco-2 intestinal cells altered tight junction structure and function, which may be a response to metabolic dysfunction.     

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells

3,4-dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [(14)C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4-methoxyphenylethanol, the product of catechol-O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K(m) value of 40 microM has been calculated.     

Toxicity of the Alternaria metabolites alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in the chicken embryo assay.

The effects in the chicken embryo assay of four Alternaria metabolites (alternariol [AOH], alternariol methyl ether [AME], altenuene [ALT], and tenuazonic acid [TA]) were investigated. Administered to 7-day-old chicken embryos by yolk sac injection, AOH, AME, and ALT caused no mortality or teratogenic effect at doses up to 1,000, 500, and 1,000 micrograms per egg, respectively. TA exhibited a calculated 50% lethal dose of 548 micrograms per egg, with no teratogenic effect observed at either lethal or sublethal doses.     

Precise determination of the <Emphasis Type="Italic">Alternaria</Emphasis> mycotoxins alternariol and alternariol monomethyl ether in cereal,fruit and vegetable products using stable isotope dilution assays

Cereal, fruit and vegetable products were analyzed for contamination with the Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) using stable isotope dilution assays (SIDAs). Both toxins were practically not detected in cereals and cereal products: AOH—one out of 13 samples at a content of 4.1 μg/kg; AME—two out of 13 samples at contents ranging between 0.2 and 0.6 μg/kg. However, if cereals for animal nutrition were analyzed, much higher values were found: AOH—five out of six samples (13–250 μg/kg); AME—six out of six samples (3–100 μg/kg). This finding may pose a potential problem concerning animal health. AOH and AME were frequently detected in vegetable products: AOH—5 out of 10 samples (2.6–25 μg/kg); AME—6 out of 10 samples (0.1–5 μg/kg). Tomato products were affected, especially. The highest content of AOH (25 μg/kg) and AME (5 μg/kg) were found in triple concentrated tomato paste. Special wines like “Trockenbeerenauslese” or “Spätlese” (affected by noble rot in the vineyard) contained AOH (4/6 samples; 1.2–4.9 μg/kg) and AME (4/6 samples; 0.1–0.3 μg/kg), but the values did not exceed the values of both toxins that were found generally in wines.     

Toxicity of the Alternaria metabolites alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in the chicken embryo assay

The effects in the chicken embryo assay of four Alternaria metabolites (alternariol [AOH], alternariol methyl ether [AME], altenuene [ALT], and tenuazonic acid [TA]) were investigated. Administered to 7-day-old chicken embryos by yolk sac injection, AOH, AME, and ALT caused no mortality or teratogenic effect at doses up to 1,000, 500, and 1,000 micrograms per egg, respectively. TA exhibited a calculated 50% lethal dose of 548 micrograms per egg, with no teratogenic effect observed at either lethal or sublethal doses.     

Microbial metabolism of 2-methyl amino acids

Eighty-nine microorganisms were isolated that were able to use 2-methyl amino acids and related compounds as the sole source of nitrogen. All of these cultures produced low levels of ammonia in culture supernatant solutions None was capable of fixing nitrogen gas. Whole-cell and cell-free-extract experiments showed that ammonia was not released directly from the 2-methyl amino acids. All of these strains except those isolated with 2-methylserine as a nitrogen source appeared to metabolize 2-methyl amino compounds by a single enzymatic reaction involving simultaneous decarboxylation and transamination. Pyruvate served as an acceptor for the transamination with the concomitant formation of alanine. The strains utilizing 2-methylserine produced a specific 2-methylserine transhydroxymethylase.     

高效液相色谱法测定大黄素在Caco-2细胞中的摄取

目的:采用反相高效液相色谱法,观察大黄素在Caco-2细胞中的摄取特点。方法:将大黄素与Caco-2细胞共同孵育,收集细胞样品,液氮反复冻融。取细胞裂解液,加入甲醇提取,提取液采用HPLC进行分析。色谱分析柱为C18柱(250mm×4．6mm,5μm,Diamonsil),流动相组成为85%乙腈及15%水(含0．1%乙酸),流速1ml·min-1,进样量20μl,柱温25℃,3D模式采集数据。结果:检测Caco-2细胞中大黄素的工作曲线的回归方程为Y=0．278x 0．148(Y=0．9996,n=5),线性范围为0．037～4．8μmol·L-1,最低检测浓度为0．018μmol·L-1。当细胞中大黄素的浓度为0．05、2和8．5μg·ml-1时,回收率分别为(101．3±7．3)%、(96．7±3．0)%和(98．7±2．1)%(n=5);相应的日内标准偏差分别为0．25%、2．9%和1．4%;相应的日间标准偏差分别为2．3%、5．6%和6．3%。大黄素在Caco-2细胞中的摄取达峰时间为10分钟,峰浓度为108．56±11．57 nmol/L·mg·protein,10分钟后Caco-2细胞中大黄素的含量迅速下降。浓度处于2-50μM之间时,Caco-2细胞对大黄素的摄取量呈线性增加,浓度达50μM后,随着剂量的增加大黄素的摄取量变化不明显。结论:大黄素可被Caco-2细胞迅速摄取,随着剂量的增加,大黄素在Caco-2细胞中的摄取存在饱和现象。     

Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 2% of cytosolic proteins) in the small intestine epithelium. Despite cell transfection studies, its function is still unclear. Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported. Furthermore, a decrease in fatty acid incorporation has been unexpectedly obtained when I-FABP reached 0. 72% of cytosolic proteins in fibroblasts (Prows et al. 1997. Arch. Biochem. Biophys. 340: 135). In the present study, the effect of a high level of I-FABP similar to amounts present in the small intestine was investigated in the human colon adenocarcinoma cell line, Caco-2. After transfection with human I-FABP cDNA, a clone expressing 1.5% I-FABP and unchanged level of liver FABP was selected. These cells, which had a lower rate of proliferation as compared with mock-transfected cells, developed the typical morphological characteristics of differentiated enterocytes. Incubation of differentiated cells with [(14)C]palmitate showed a 34% reduction (P < 0.01) of fatty acid incorporation, whereas the relative distribution of radiolabel into triglycerides was not affected. A nonsignificant 21% reduction of fatty acid incorporation was observed with another clone expressing 10-fold less I-FABP. In conclusion, a high level of I-FABP expressed in a differentiated enterocyte model inhibited fatty acid incorporation, by a mechanism which remains to be defined.