Identification of ochratoxin-N-acetyl-L-cysteine as a new ochratoxin A metabolite and potential biomarker in human urine.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin with nephrocarcinogenic potential found in a broad spectrum of food commodities. The mode of action of this compound, as well as its metabolism, is still not fully understood. To determine whether the conjugation of OTA with glutathione plays an important role in human OTA metabolism, an ochratoxin-glutathione conjugate (OTB-GSH), as well as the corresponding urinary metabolite ochratoxin-N-acetyl-L-cysteine (OTB-NAC), were synthesized and their structures confirmed by NMR spectroscopy. By means of synthesized stable isotope-labeled d5-OTB-GSH and d5-OTB-NAC references, a sensitive HPLC-MS/MS method has been developed and applied for the screening of human urine samples. OTB-NAC could be detected in 11 of the analyzed 18 urine samples and was quantified in 5 urine samples in the range between 0.023 and 0.176 ng mg-1 creatinine. OTB-GSH has not been detected in the urine samples. In OTB-NAC positive samples, this metabolite contributed to a comparable concentration range to the total OTA excretion as the parent compound. This is the first direct analysis of an OTA phase 2 metabolite in humans.

Human Study on the Kinetics of 2'R-Ochratoxin A in the Blood of Coffee Drinkers.

SCOPE: The aim of this study is to obtain a deeper knowledge of the kinetics of 2'R-ochratoxin A (2'R-OTA), the thermal degradation product of the mycotoxin ochratoxin A (OTA). To investigate the correlation between the amount of this compound in roasted coffee and human blood samples, a human study is performed. METHODS AND RESULTS: An 18-week human study is carried out. During the first eight weeks, all known 2'R-OTA-containing food sources are excluded from the diet and the reduction of 2'R-OTA in venous blood is analyzed. Afterwards, participants are allowed to consume coffee with known OTA and 2'R-OTA concentrations. On a biweekly scale, 2'R-OTA and OTA blood levels are determined. After eight weeks of fasting on 2'R-OTA-containing foods, the 2'R-OTA blood concentration decreased by about 10%. Based on this, a long biological half-life of over seven months is estimated. In the 24 h urine samples collected before and after the coffee fasting period, only traces of 2'R-OTA are detected. CONCLUSION: Results show that 2'R-OTA has a more than seven-fold higher biological half-life in human blood compared to OTA (approx. 35 days). The reason for the long persistence of 2'R-OTA in human blood is still unclear and further research is needed.

Occurrence of the Ochratoxin A Degradation Product 2'R-Ochratoxin A in Coffee and Other Food: An Update.

Food raw materials can contain the mycotoxin ochratoxin A (OTA). Thermal processing of these materials may result in decreased OTA levels but also in the formation of the thermal isomerization product 2'R-ochratoxin A (2'R-OTA). So far, only 2'R-OTA levels reported from 15 coffee samples in 2008 are known, which is little when compared to the importance of coffee as a food and trading good. Herein, we present results from a set of model experiments studying the effect of temperatures between 120 °C and 270 °C on the isomerization of OTA to 2'R-OTA. It is shown that isomerization of OTA starts at temperatures as low as 120 °C. At 210 °C and above, the formation of 25% 2'R-OTA is observed in less than one minute. Furthermore, 51 coffee samples from France, Germany, and Guatemala were analyzed by HPLC-MS/MS for the presence of OTA and 2'R-OTA. OTA was quantified in 96% of the samples, while 2'R-OTA was quantifiable in 35% of the samples. The highest OTA and 2'R-OTA levels of 28.4 µg/kg and 3.9 µg/kg, respectively, were detected in coffee from Guatemala. The OTA:2'R-OTA ratio in the samples ranged between 2.5:1 and 10:1 and was on average 5.5:1. Besides coffee, 2'R-OTA was also for the first time detected in a bread sample and malt coffee powder.

Interaction of the mycotoxin metabolite dihydrocitrinone with serum albumin.

Citrinin (CIT) is a nephrotoxic mycotoxin produced by Penicillium, Monascus, and Aspergillus species. CIT appears as a contaminant in cereals, cereal-based products, fruits, nuts, and spices. During the biotransformation of CIT, its major urinary metabolite dihydrocitrinone (DHC) is formed. Albumin interacts with several compounds (including mycotoxins) affecting their tissue distribution and elimination. CIT-albumin interaction is known; however, the complex formation of DHC with albumin has not been reported previously. In this study, we aimed to investigate the interaction of DHC with albumin, employing fluorescence spectroscopy, circular dichroism, and molecular modeling studies. Furthermore, species differences and thermodynamics of the interaction as well as the effects of albumin on the acute in vitro toxicity of DHC and CIT were also tested. Our main observations/conclusions are as follows: (1) Fluorescence signal of DHC is strongly enhanced by albumin. (2) Formation of DHC-albumin complexes is supported by both fluorescence spectroscopic and circular dichroism studies. (3) DHC forms similarly stable complexes with human albumin (K~105 L/mol) as CIT. (4) DHC-albumin interaction did not show significant species differences (tested with human, bovine, porcine, and rat albumins). (5) Based on modeling studies and investigations with site markers, DHC occupies the Heme binding site (subdomain IB) on human albumin. (6) The presence of albumin significantly decreased the acute in vitro cytotoxic effects of both DHC and CIT on MDCK cell line.

Interaction of 2'R-ochratoxin A with Serum Albumins: Binding Site, Effects of Site Markers, Thermodynamics, Species Differences of Albumin-binding, and Influence of Albumin on Its Toxicity in MDCK Cells.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2'R-ochratoxin A (2'R-OTA), which appears in the blood of coffee drinkers. Human serum albumin (HSA) binds 2'R-OTA (and OTA) with high affinity; therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2'R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2'R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2'R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2'R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2'R-OTA than OTA from HSA. Fluorescence and binding constants of 2'R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2'R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA.

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus. 2′R-Ochratoxin A (2′R-OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′R-OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′R-OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′R-OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′R-OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a logK of 7.0⁻7.6 was calculated, while for its thermally produced isomer 2′R-OTA, a lower, but still high, logK of 6.2⁻6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′R-OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′R-OTA found in human blood samples.