Oxidative metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in precision-cut rat liver slices in vitro

Scope: Monohydroxylation of alternariol (AOH) and alternariol‐9‐methyl ether (AME) has previously been reported as a prominent metabolic route under cell‐free conditions. This pathway gives rise to several catechol metabolites and may therefore be of toxicological relevance. Methods and results: To clarify whether hydroxylation of AOH and AME occurs under in vivo‐like conditions in the presence of conjugation reactions, the metabolism of the Alternaria toxins has now been studied in precision‐cut rat liver slices. Four catechol metabolites of AOH and two of AME, together with several of their O‐methylation products, as catalyzed by catechol‐O‐methyl transferase, were clearly identified after incubation of the liver slices with AOH and AME. These metabolites were predominantly present as conjugates with glucuronic acid and/or sulfate. In preliminary studies with bile duct‐cannulated male rats dosed with AOH by gavage, the four monohydroxylated metabolites of AOH could also be demonstrated in the bile either as catechols or as O‐methyl ethers. Conclusion: These experiments clearly show that AOH and AME undergo catechol formation in vivo and warrant closer examination of the toxicological significance of this metabolic pathway.     

Glucuronidation of zearalenone,zeranol and four metabolites in vitro: Formation of glucuronides by various microsomes and human UDP‐glucuronosyltransferase isoforms

Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α‐zearalanol (α‐ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α‐ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP‐glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14‐hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α‐ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.     

Aerobic and anaerobic in vitro testing of feed additives claiming to detoxify deoxynivalenol and zearalenone

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins produced by fungi of the genus Fusarium which frequently contaminate maize and grain cereals. Mycotoxin-contaminated feed endangers animal health and leads to economic losses in animal production. Several mycotoxin elimination strategies, including the use of commercially available DON and ZEN detoxifying agents, have been developed. However, frequently there is no scientific proof of the efficacy of such adsorbents and degrading products. We therefore tested 20 commercially available products claiming to detoxify DON and/or ZEN either by biodegradation (4 products) or a combination of degradation and adsorption (16 products) under aerobic and anaerobic conditions at approx. pH 7. Under the applied conditions, a complete reduction of DON and consequent formation of the known non-toxic metabolite DOM-1 was exclusively observed in samples taken from the anaerobic degradation experiment of one product. For all other products, incubated under aerobic and anaerobic conditions, a maximum DON reduction of 17% after 72 h of incubation was detected. Aerobic and anaerobic incubation of only one tested product resulted in complete ZEN reduction as well as in the formation of the less-toxic metabolites DHZEN and HZEN. With this product, 68–97% of the toxin was metabolised within 3 h. After 24 h, a ZEN reduction ≥ 60% was obtained with four additional products during aerobic incubation only. Six of the 20 investigated products produced α- and/or β-ZEL, which are metabolites showing similar oestrogenic activity compared to ZEN. Aerobic and anaerobic degradation to unknown metabolites with unidentified toxicity was obtained with 10 and 3 products, respectively. The results of our study demonstrate the importance of in vitro experiments to critically screen agents claiming mycotoxin detoxification.     

Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro

Scope: Zearalenone (ZEN) and α‐zearalanol (α‐ZAL, zeranol) were studied in differentiated Caco‐2 cells and in the Caco‐2 Millicell^® system in vitro to simulate their in vivo intestinal absorption and metabolism in humans. Methods and results: In addition to metabolic reduction/oxidation, extensive conjugation with glucuronic acid and sulfate of the parent compounds and their phase I metabolites was observed. The positional isomers of the glucuronides and sulfates were unambiguously identified: Sulfonation occurred specifically at the 14‐hydroxyl group, whereas glucuronidation was less specific and, in addition to the preferred 14‐hydroxyl group, involved the 16‐ and 7‐hydroxyl groups. Using the Caco‐2 Millicell^® system, an efficient transfer of the glucuronides and sulfates of ZEN and α‐ZAL and their phase I metabolites into both the basolateral and the apical compartment was observed after apical administration. The apparent permeability coefficients (P_app values) of ZEN, α‐ZAL and the ZEN metabolite α‐zearalenol were determined, using an initial apical concentration of 20 μM and a permeation time of 1 h. Conclusion: According to the P_app values, the three compounds are expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycones and as glucuronide and sulfate conjugates in humans.     

Novel oxidative metabolites of the mycoestrogen zearalenone in vitro

The estrogenic mycotoxin zearalenone (ZEN) is known to get metabolized to the alpha-and beta-isomers of zearalenol, but no hydroxylation products of ZEN have yet been reported as metabolites in animals or humans. We have therefore incubated ZEN with microsomes from rat liver in the presence of a nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-regenerating system and analyzed the extracted metabolites with HPLC and GC-MS after trimethylsilylation. A total of 17 in vitro metabolites were observed. The two major metabolites were tentatively identified as monohydroxylated ZEN with the newly introduced hydroxyl group localized in the aliphatic macrocyclic ring. According to the GC-MS analysis, other six monohydroxylation products of ZEN were formed as minor metabolites, together with alpha-and beta-zearalenol and monohydroxylated zearalenols. Thus, ZEN has a considerable propensity for undergoing metabolic hydroxylation reactions in vitro, and the in vivo formation and biological properties of such oxidative metabolites should now be studied.     

The C‐glycosyl flavonoid,aspalathin, is absorbed,methylated and glucuronidated intact in humans

Human bioavailability of the flavonoid dihydrochalcones is little understood, and no evidence exists for C‐glycosyl flavonoid absorption in humans. The present study uses catechol‐O‐methyltransferase to generate methylated metabolites of aspalathin (a C‐glycosyl dihydrochalcone from rooibos tea). One of the methylated forms, both with and without glucuronidation, was detected using LC‐MS/MS in the urine of human subjects (n = 6), demonstrating that deglycosylation is not a prerequisite for C‐glycosyl flavonoid absorption. Methylation is catalysed by both intestine and liver cytosolic extracts. The results show that flavonoid C‐glycosides are methylated and glucuronidated in vivo in an intact form in humans.     

In vitro anti-platelet effects of simple plant-derived phenolic compounds are only found at high, non-physiological concentrations

Scope : Bioactive polyphenols from fruits, vegetables, and beverages have anti‐platelet effects and may thus affect the development of cardiovascular disease. We screened the effects of 26 low molecular weight phenolic compounds on two in vitro measures of human platelet function. Methods and results : After platelets had been incubated with one of 26 low molecular weight phenolic compounds in vitro, collagen‐induced human platelet aggregation and in vitro TRAP‐induced P‐selectin expression (as marker of platelet activation) were assessed. Incubation of platelet‐rich plasma from healthy volunteers with 100 μmol/L hippuric acid, pyrogallol, catechol, or resorcinol significantly inhibited collagen‐induced platelet aggregation (all p<0.05; n≥15). Incubation of whole blood with concentrations of 100 μmol/L salicylic acid, p‐coumaric acid, caffeic acid, ferulic acid, 4‐hydroxyphenylpropionyl glycine, 5‐methoxysalicylic acid, and catechol significantly inhibited TRAP‐induced surface P‐selectin expression (all p<0.05; n=10). Incubation with lower concentrations of phenolics affected neither platelet aggregation nor activation. Conclusion : As concentrations of 100 μmol/L are unlikely to be reached in the circulation, it is doubtful whether consumption of dietary phenolics in nutritionally attainable amounts plays a major role in inhibition of platelet activation and aggregation in humans.     

Effects of Catechol O‐Methyl Transferase Inhibition on Anti‐Inflammatory Activity of Luteolin Metabolites

Although luteolin is known to have potent anti‐inflammatory activities, much less information has been provided on such activities of its hepatic metabolites. Luteolin was subjected to hepatic metabolism in HepG2 cells either without or with catechol O‐methyl transferase (COMT) inhibitor. To identify hepatic metabolites of luteolin without (luteolin metabolites, LMs) or with COMT inhibitor (LMs+CI), metabolites were treated by β‐glucuronidase and sulfatase, and found that they were composed of glucuronide and sulfate conjugates of diosmetin in LMs or these conjugates of luteolin in LMs+CI. LMs and LMs+CI were examined for their anti‐inflammatory activities on LPS stimulated Raw 264.7 cells. Expression of iNOS and production of nitric oxide and pro‐inflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 were suppressed more effectively by the treatment with LMs+CI than LMs. Our data provide a new insight on possible improvement in functional properties of luteolin on target cells by modifying their metabolic pathway in hepatocytes.     

Detoxification of zearalenone and ochratoxin A by ozone and quality evaluation of ozonised corn

Zearalenone (ZEN) and ochratoxin A (OTA) are secondary toxic metabolites of fungi that can contaminate a wide range of food and feedstuff. In this study, the effects of ozone treatment on ZEN and OTA and the quality of ozonised corn are investigated. Ozone significantly affects ZEN and OTA solutions. ZEN was undetectable 5 s after being treated with 10 mg l^–1 ozone. However, OTA was resistant to ozonation with a degradation rate of 65.4% after 120 s of treatment. Moreover, ZEN and OTA solutions were difficult to degrade after being dried by a nitrogen stream. Results showed that ozone effectively degraded ZEN and OTA in corn. The degradation rates of ZEN and OTA in corn increased with ozone concentration and treatment time. The degradation of ZEN and OTA at different ozone concentrations appropriately conformed to first-order kinetics with an R² value > 0.8749. Furthermore, under the same conditions, corn with increased moisture content (MC) (19.6%) was more sensitive to ozone than corn with a low MC (14.1%). When treated with 100 mg l^–1 ozone for 180 min, ZEN and OTA in corn with 19.6% MC decreased by 90.7% and 70.7%, respectively. To evaluate the quality of ozonised corn, subsequent quality experiments were conducted using corn samples treated at different times with 100 mg l^–1 ozone. The MC of corn decreased after ozone treatment. The whiteness and yellowness of the corn increased and decreased with increasing time, respectively. The fatty acid value of the corn increased significantly (p ≤ 0.05) after 180 min of treatment. This study verified that ozone can effectively degrade ZEN and OTA in corn, but slightly affected corn quality.     

Recovery and metabolism of xanthohumol in germ‐free and human microbiota‐associated rats

The impact of human intestinal bacteria on the bioavailability of the prenylflavonoid xanthohumol (XN) was studied by comparing germ‐free (GF) and human microbiota‐associated (HMA) rats. After XN application, XN, XN conjugates, and isoxanthohumol (IX) conjugates occurred in blood samples of GF and HMA rats, whereas IX was detected only in the blood of HMA rats. Overall excretion of XN and its metabolites within 48 h was only 4.6% of the ingested dose in GF rats and 4.2% in HMA rats, feces being the major route of excretion. While both GF and HMA rats excreted XN, IX, and their conjugates with urine and feces, 8‐prenylnaringenin and its corresponding conjugates were exclusively observed in the feces of HMA rats. The microbial formation of 8‐prenylnaringenin was confirmed by incubation of XN and IX with human fecal slurries. The amount of conjugates excreted in urine and feces was lower in HMA rats compared to GF rats indicating their hydrolysis by human intestinal microbiota. Thus, the impact of bacteria on the XN metabolism in the gut may affect the in vivo effects of ingested XN.     

Simultaneous detection of deoxynivalenol and zearalenone by dual-label time-resolved fluorescence immunoassay

BACKGROUND: The health risks of deoxynivalenol (DON) and zearalenone (ZEN) necessitate the development of analytical methods for widespread food and feed screening. We sought to establish a rapid, economic and sensitive dual‐label time‐resolved fluoroimmunoassay (TRFIA) to detect DON and ZEN simultaneously. Eu³⁺‐ and Sm³⁺‐labelled antibodies were used, as lanthanides are more stable and have narrower emission spectra than most fluorescent dyes. RESULTS: The limit of detection was 0.0194 ng mL^?1 for DON (range: 0.0194–100 ng mL^?1) and 0.37 ng mL^?1 for ZEN (range: 0.37–50 ng mL^?1). DON recovery in spiked cereal samples was 88–107%, and for ZEN was 83–108%, with both intra‐ and inter‐assay coefficients of variation (CVs) less than 5%. The dual‐label TRFIA results correlated well with ELISA results (correlation coefficients: 0.9733 for DON and 0.9784 for ZEN). CONCLUSION: The dual‐label TRFIA is a simple, fast and sensitive method for high‐throughput screening of DON and ZEN in food and feedstuff. Copyright © 2010 Society of Chemical Industry     

Zearalenone: Incidence,toxigenic fungi and chemical decontamination in Egyptian cereals

An investigation was made for the occurrence of Zearalenone (ZEN) and Zearalenone producing fungi in cereals (corn, 50 samples; rice, 45 samples; wheat, 40 samples) collected from Egypt. ZEN was detected in 15 of 50 corn samples with an average concentration of 22.3 ppb. The incidence of ZEN in rice samples was 8.9% (4 samples of 45), and the average was 15.5 ppb. Out of 40 wheat samples 5 samples were contaminated with ZEN (12.5%) with an average of 8.8 ppb. Seventy-nine Fusarium strains belonging to 9 different species were isolated from Egyptian cereals, and when tested for ZEN production only 26 isolates were Zearalenone producer. Efficiency of H₂O₂ for destruction of ZEN in contaminated corn was studied at different concentration (3, 5 and 10%). The results revealed that per cent of disappearance of ZEN was found to be dependent upon the concentration of H₂O₂, temperature and period of exposure. The highest per cent of degradation was 83.9%, with 10% H₂O₂ at 80 °C for 16 h, followed by 75% at the same conditions for 8 h, while the lowest one obtained by 3% H₂O₂ at 50 °C for 2 h.     

Bioavailability study of a polyphenol‐enriched extract from Hibiscus sabdariffa in rats and associated antioxidant status

The aqueous extracts of Hibiscus sabdariffa have been commonly used in folk medicine. Nevertheless, the compounds or metabolites responsible for its healthy effects have not yet been identified. The major metabolites present in rat plasma after acute ingestion of a polyphenol‐enriched Hibiscus sabdariffa extract were characterized and quantified in order to study their bioavailability. The antioxidant status of the plasma samples was also measured through several complementary antioxidant techniques. High‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry (HPLC‐ESI‐TOF‐MS) was used for the bioavailability study. The antioxidant status was measured by ferric reducing ability of plasma method, thiobarbituric acid reactive substances assay, and superoxide dismutase activity assay. Seventeen polyphenols and metabolites have been detected and quantified. Eleven of these compounds were metabolites. Although phenolic acids were found in plasma without any modification in their structures, most flavonols were found as quercetin or kaempferol glucuronide conjugates. Flavonol glucuronide conjugates, which show longer half‐life elimination values, are proposed to contribute to the observed lipid peroxidation inhibitory activity in the cellular membranes. By contrast, phenolic acids appear to exert their antioxidant activity through ferric ion reduction and superoxide scavenging at shorter times. We propose that flavonol‐conjugated forms (quercetin and kaempferol) may be the compounds responsible for the observed antioxidant effects and contribute to the healthy effects of H. sabdariffa polyphenolic extract.     

Binding affinity of tea catechins for HSA: Characterization by high‐performance affinity chromatography with immobilized albumin column

Catechins are the major polyphenols in green tea leaves. Recent studies have suggested that the catechins form complexes with HSA for transport in human blood, and their binding affinity for albumin is believed to modulate their bioavailability. In this study, the binding affinities of catechins and their analogs were evaluated and the relationship between the chemical structure of each catechin and its binding property were investigated. Comparing these catechins by HPLC analysis with the HSA column, we showed that galloylated catechins have higher binding affinities with HSA than non‐galloylated catechins. In addition, pyrogallol‐type catechins have a high affinity compared to catechol‐type catechins. Furthermore, the binding affinity of the catechin with 2,3‐trans structure was higher than those of the catechin with 2,3‐cis structure. The importance of the hydroxyl group on the galloyl group and B‐ring was confirmed using methylated catechins. These results indicate that the most important structural element contributing to HSA binding of tea catechins is the galloyl group, followed by the number of hydroxyl groups on the B‐ring and the galloyl group or the configuration at C‐2. Our findings provide fundamental information on the relationship between the chemical structure of tea catechins and its biological activity.     

Urinary Excretion of Niacin Metabolites in Humans After Coffee Consumption

Scope

Coffee is a major natural source of niacin in the human diet, as it is formed during coffee roasting from the alkaloid trigonelline. The intention of our study was to monitor the urinary excretion of niacin metabolites after coffee consumption under controlled diet.

Methods and results

We performed a 4‐day human intervention study on the excretion of major niacin metabolites in the urine of volunteers after ingestion of 500 mL regular coffee containing 34.8 μmol nicotinic acid (NA) and 0.58 μmol nicotinamide (NAM). In addition to NA and NAM, the metabolites N¹‐methylnicotinamide (NMNAM), N¹‐methyl‐2‐pyridone‐5‐carboxamide (2‐Py), and nicotinuric acid (NUA) were identified and quantified in the collected urine samples by stable isotope dilution analysis (SIVA) using HPLC‐ESI‐MS/MS. Rapid urinary excretion was observed for the main metabolites (NA, NAM, NMNAM, and 2‐Py), with t_max values within the first hour after ingestion. NUA appeared in traces even more rapidly. In sum, 972 nmol h^?1 of NA, NAM, NMNAM, and 2‐Py were excreted within 12 h after coffee consumption, corresponding to 6% of the ingested NA and NAM.

Conclusion

The results indicate regular coffee consumption to be a source of niacin in human diet.

     

A Review on Mycotoxins in Food and Feed: Malaysia Case Study

Fungi are distributed worldwide and can be found in various foods and feedstuffs from almost every part of the world. Mycotoxins are secondary metabolites produced by some fungal species and may impose food safety risks to human health. Among all mycotoxins, aflatoxins (AFs), ochratoxin A (OTA), trichothecenes, deoxynivalenol (DON and T‐2 toxin), zearalenone (ZEN), and fumonisins (FMN) have received much attention due to high frequency and severe health effects in humans and animals. Malaysia has heavy rainfall throughout the year, high temperatures (28 to 31 °C), and high relative humidity (70% to 80% during wet seasons). Stored crops under such conditions can easily be contaminated by mycotoxin‐producing fungi. The most important mycotoxins in Malaysian foods are AFs, OTA, DON, ZEN, and FMN that can be found in peanuts, cereal grains, cocoa beans, and spices. AFs have been reported to occur in several cereal grains, feeds, nuts, and nut products consumed in Malaysia. Spices, oilseeds, milk, eggs, and herbal medicines have been reported to be contaminated with AFs (lower than the Malaysian acceptable level of 35 ng/g for total AFs). OTA, a possible human carcinogen, was reported in cereal grains, nuts, and spices in Malaysian market. ZEN was detected in Malaysian rice, oat, barley, maize meal, and wheat at different levels. DON contamination, although at low levels, was reported in rice, maize, barley, oat, wheat, and wheat‐based products in Malaysia. FMN was reported in feed and some cereal grains consumed in Malaysia. Since some food commodities are more susceptible than others to fungal growth and mycotoxin contamination, more stringent prevention and control methods are required.     

Preparation of Methylated Products of A‐type Procyanidin Trimers in Cinnamon Bark and Their Protective Effects on Pancreatic β‐Cell

Polyphenols are partial metabolized to methylated conjugations in vivo, and then could modify bioavailability and bioactivity related to the uptake of parent compounds. Our previous studies have found that the antidiabetic effects of cinnamon barks are mainly related to polyphenol components, particularly A‐type procyanidin trimer cinnamtannin‐1 (CT1). It is necessary to understand the antidiabetic activity of methylations of CT1, nevertheless, sufficient amounts of methylated CT1 are difficult to obtain from metabolites in vivo. In this study, O‐methyl derivatives of CT1 were prepared through one‐pot methyl iodide reaction and isolation via column chromatography and RP‐HPLC semipreparation. The structures of O‐methyl substituents were determined through NMR (Nuclear Magnetic Resonance) and HPLC‐ESI‐MS (High‐performance liquid chromatography‐electrospray ionization‐mass spectrometry). Five purified O‐methyl substituents and 2 isomers of CT1 were obtained. Their protective effects on a palmitic acid‐induced pancreatic β‐cell apoptosis model were then evaluated. Results showed that the protective effects on pancreatic β‐cell of O‐methyl substituents were weaker than those of CT1. The results suggested that the methylation of catechol groups could be a relevant factor contributing to the decline of protective effects on pancreatic β‐cell of CT1 via obstructing quinone intermediate formation and affecting antioxidant abilities. The antidiabetic effects of O‐methyl derivatives of CT1 should be further determined by other antidiabetic models.     

The red clover isoflavone irilone is largely resistant to degradation by the human gut microbiota

Intestinal bacteria may influence bioavailability and physiological activity of dietary isoflavones. We therefore investigated the ability of human intestinal microbiota to convert irilone and genistein in vitro. In contrast to genistein, irilone was largely resistant to transformation by fecal slurries of ten human subjects. The fecal microbiota converted genistein to dihydrogenistein, 6′‐hydroxy‐O‐desmethylangolensin, and 2‐(4‐hydroxyphenyl)‐propionic acid. However, considerable interindividual differences in the rate of genistein degradation and the pattern of metabolites formed from genistein were observed. Only one metabolite, namely dihydroirilone, was formed from irilone in minor amounts. In further experiments, Eubacterium ramulus, a prevalent flavonoid‐degrading species of the human gut, was tested for transformation of irilone. In contrast to genistein, irilone was not converted by E. ramulus. Irilone only differs from genistein by a methylenedioxy group attached to the A‐ring of the isoflavone skeleton. This substitution obviously restricts the degradability of irilone by human intestinal bacteria.     

Inhibition of polyphenol oxidase obtained from various sources by 2,3‐diaminopropionic acid

This paper reports for the first time the inhibition of the catecholase activities of mushroom, artichoke (Cynara scolymus L) and Ocimum basilicum L polyphenol oxidase by 2,3‐diaminopropionic acid. Polyphenol oxidases from artichoke and O basilicum L were purified by ammonium sulfate precipitation, dialysis and a Sepharose 4B‐L ‐tyrosine‐p‐aminobenzoic acid‐affinity column. In inhibition studies, 2,3‐diaminopropionic acid showed uncompetitive inhibition for mushroom PPO using catechol and pyrogallol as substrates, competitive inhibition for O basilicum L PPO using catechol as a substrate, and uncompetitive inhibition for artichoke PPO using catechol as a substrate. Furthermore, sodium azide, which is an inhibitor of PPO, was used as an inhibitor for comparison with the inhibition potency of 2,3‐diaminopropionic acid. The highest 2,3‐diaminopropionic acid inhibition observed with O basilicum L (K_i = 0.89 mM ), followed by artichoke (K_i = 1.42 mM ) and mushroom (K_i = 2.47 mM ), respectively. Copyright © 2005 Society of Chemical Industry