The Possible Role of Mycotoxins in the Pathogenesis of Endometrial Cancer.

Endometrial cancer is one of the most common cancer types among women. Many factors can contribute to the development of this disease, including environmental factors and, thus, eating habits. Our study aims to determine the levels of various mycotoxins and their metabolites in the blood serum and endometrial tissue samples of participants with previously proven endometrial cancer and to find possible contributions to cancer development. In the cohort clinical trial, 52 participants aged between 44 and 86 were studied. The participants were divided into two groups: patients or matched controls. All patients had previously histologically diagnosed endometrial cancer. The cancer patients were divided into low-grade endometrioid and low- plus high-grade endometrioid groups. Controls had no history of endometrial malignancy or premalignancy. Blood serum and endometrial tissue samples were obtained from all study patients. We compared the concentrations of total Aflatoxins (Afs), Deoxynivalenol (DON), Ochratoxin-A (OTA), T2-toxin and HT2 toxin (T2/HT2 toxin), Zearalenone (ZEN), alpha-Zearalenol (α-ZOL), and Fumonisin B1 (FB1) in the serum and endometrium between the different study groups. As a result, we can see a significant correlation between the higher levels of Afs and zearalenone and the presence of endometrial cancer. In the case of Afs, DON, OTA, T2/HT2 toxins, ZEN, and alpha-ZOL, we measured higher endometrial concentrations than in serum. Considering the effect of mycotoxins and eating habits on cancer development, our results might lead to further research exploring the relationship between certain mycotoxins and endometrium cancer.

Assessment of Mycotoxin Exposure and Associated Risk in Pregnant Dutch Women: The Human Biomonitoring Approach.

Mycotoxins are toxic secondary metabolites produced by various fungi that can contaminate food crops, which, in turn, may lead to human exposure. Chronic exposure to mycotoxins can cause adverse health effects including reproductive and developmental toxicity. Pregnant women and their foetuses present a vulnerable group for exposure to mycotoxins that can cross the placenta. Human biomonitoring of mycotoxins provides a real-life approach to estimate internal exposure. In this pilot study, 24-h urine samples from 36 pregnant Dutch women were analysed for aflatoxin M1 (AFM1), total deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM-1), total zearalenone (ZEN), total α-zearalenol (α-ZEL), total ß-zearalenol (ß-ZEL) and total zearalanone (ZAN), where 'total' refers to mycotoxins and their conjugated forms. Serum samples from these women were analysed for fumonisin B1 (FB1) and ochratoxin A (OTA). All samples were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most prevalent mycotoxins were total DON, total ZEN and OTA, with a detection frequency of 100%. DOM-1, total α-ZEL and total ß-ZEL were detected but to a lesser extent, while AFM1, total ZAN and FB1 were undetected. Median concentrations were 4.75 µg total DON/L, 0.0350 µg DOM-1/L, 0.0413 µg total ZEN/L, 0.0379 µg total α-ZEL/L, 0.0189 µg total ß-ZEL/L, and 0.121 µg OTA/L. The calculated median concentration for total ZEN and its metabolites was 0.105 µg/L. Based on two separate risk assessment approaches, total DON exposure in this group was considered to be of low concern. Similarly, exposure to total ZEN and its metabolites in this group was of low concern. For OTA, the risk of non-neoplastic effects was of low concern based on exposure in this group, and the risk of neoplastic effects was of low concern in the majority of participants in this group. The findings of this pilot study confirm the presence of mycotoxins in the urine and serum of pregnant Dutch women, with total DON, total ZEN, and OTA most frequently detected. Exposure to all measured mycotoxins was considered to be of low concern in this group, except for exposure to OTA, which was of low concern for the majority of participants. The study's findings offer valuable insights but should be confirmed using a larger and more diverse sample of the Dutch general population.

The screening method for 39 phytotoxins and mycotoxins in blood and urine with liquid chromatography-high resolution mass spectrometry.

BACKGROUND: Poisonings caused by plant toxins and mycotoxins occur frequently, which do great harm to human health and social public health safety. When a poisoning incident occurs, biological samples are commonly be used to conduct the detection of toxic substances and their metabolites for targeted clinical treatment and incident analysis. OBJECTIVE: To establish an efficient and accurate analysis method of 39 phytotoxins and mycotoxins in blood and urine by high performance liquid chromatography quadrupole tandem orbitrap mass spectrometry (HPLC-Orbitrap MS). METHOD: After 3 mL of methanol being added to 1 mL blood and urine respectively for extraction and protein precipitation, the supernatant was injected into HPLC-Orbitrap MS for analysis. The phytotoxins and mycotoxins were separated by Hypersil GOLD PFP column with gradient elution using methanol-5 mmol/L ammonium acetate as mobile phase. The data were collected in ESI positive ion mode using Full MS/dd-MS2 for mass spectrometry detection. RESULT: The mass database of 39 phytotoxins and mycotoxins was developed, and accurate qualitative analysis can be obtained by matching with the database using the proposed identification criteria. Limit of detections (LODs) were 1.34 × 10-4 âˆ¼ 1.92 ng/mL and 1.92 × 10-4 âˆ¼ 9.80 ng/mL for blood and urine samples, respectively. Limits of quantification (LOQ) of toxins in blood and urine ranged from 4.47 × 10-4 âˆ¼ 6.32 ng/mL and 6.39 × 10-4 âˆ¼ 32.67 ng/mL, respectively. Intra-day relative standard deviations (RSDs) were 0.79 % âˆ¼ 10.90 %, and inter-day RSDs were 1.08 % âˆ¼ 18.93 %. The recoveries can reach 90 % âˆ¼ 110 % with matrix matching calibration curves. CONCLUSION: The established method is simple and rapid to operate, which can complete the sample analysis within 30 min, providing technical support for clinical poisoning treatment and public health poisoning analysis.

Biomonitoring of Mycotoxins in Plasma of Patients with Alzheimer's and Parkinson's Disease.

Exposure to environmental contaminants might play an important role in neurodegenerative disease pathogenesis, such as Parkinson´s disease (PD) and Alzheimer´s disease (AD). For the first time in Spain, the plasmatic levels of 19 mycotoxins from patients diagnosed with a neurodegenerative disease (44 PD and 24 AD) and from their healthy companions (25) from La Rioja region were analyzed. The studied mycotoxins were aflatoxins B1, B2, G1, G2 and M1, T-2 and HT-2, ochratoxins A (OTA) and B (OTB), zearalenone, sterigmatocystin (STER), nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deepoxy-deoxynivalenol, neosolaniol, diacetoxyscirpenol and fusarenon-X. Samples were analyzed by LC-MS/MS before and after treatment with ß-glucuronidase/arylsulfatase in order to detect potential metabolites. Only OTA, OTB and STER were detected in the samples. OTA was present before (77% of the samples) and after (89%) the enzymatic treatment, while OTB was only detectable before (13%). Statistically significant differences in OTA between healthy companions and patients were observed but the observed differences might seem more related to gender (OTA levels higher in men, p-value = 0.0014) than the disease itself. STER appeared only after enzymatic treatment (88%). Statistical analysis on STER, showed distributions always different between healthy controls and patients (patients' group > controls, p-value < 0.0001). Surprisingly, STER levels weakly correlated positively with age in women (rho = 0.3384), while OTA correlation showed a decrease of levels with age especially in the men with PD (rho = -0.4643).

Mycotoxin Biomarkers in Pigs-Current State of Knowledge and Analytics.

Farm animals are frequently exposed to mycotoxins, which have many adverse effects on their health and become a significant food safety issue. Pigs are highly exposed and particularly susceptible to mycotoxins, which can cause many adverse effects. For the above reasons, an appropriate diagnostic tool is needed to monitor pig' exposure to mycotoxins. The most popular tool is feed analysis, which has some disadvantages, e.g., it does not include individual exposure. In recent years, the determination of biomarkers as a method to assess the exposure to mycotoxins by using concentrations of the parent compounds and/or metabolites in biological matrices is becoming more and more popular. This review provides a comprehensive overview of reported in vivo mycotoxin absorption, distribution, metabolism and excretion (ADME) and toxicokinetic studies on pigs. Biomarkers of exposure for aflatoxins, deoxynivalenol, ochratoxin A, fumonisins, T-2 toxin and zearalenone are described to select the most promising compound for analysis of porcine plasma, urine and faeces. Biomarkers occur in biological matrices at trace levels, so a very sensitive technique-tandem mass spectrometry-is commonly used for multiple biomarkers quantification. However, the sample preparation for multi-mycotoxin methods remains a challenge. Therefore, a summary of different biological samples preparation strategies is included in that paper.

Evaluation of Inner Exposure of Horses to Zearalenone (ZEN), Deoxynivalenol (DON) and Their Metabolites in Relation to Colic and Health-Related Clinical-Chemical Traits.

Mycotoxin contaminated feed has been associated with colic of horses caused by intestinal disorders. Whether such disease conditions alter the intestinal toxin metabolism and transfer across a compromised mucosal barrier is unknown. A screening approach was used to relate blood residue levels of DON, ZEN and their metabolites to the status of the horses (sick vs. healthy). A total of 55 clinically healthy horses from 6 different farms with varying feeding background served as control for sick horses (N = 102) hospitalized due to colic. ZEN, alpha-zearalenol (ZEL), beta-ZEL and DON were detectable in peripheral blood as indicators for the inner exposure with significant farm effects for alpha- and beta-ZEL. However, the levels in sick horses were similar to all farms. Moreover, the proportion of beta-ZEL of all detected ZEN metabolites as an indicator for the degree of metabolism of ZEN was not different for sick horses but differed amongst the control farms. Although the incidence of DON in blood was generally low and not significantly different amongst healthy and sick horses, the positive samples were nearly exclusively found in sick horses suggesting either a higher toxin transfer, an association of DON with the development of colic or a different feeding background.

Volumetric Absorptive Microsampling as an Alternative Tool for Biomonitoring of Multi-Mycotoxin Exposure in Resource-Limited Areas.

Biomonitoring of biological samples arises as an effective tool to evaluate the exposure to mycotoxins in the population. Owing to the wide range of advantages, there is a growing interest in the use of non- and minimally invasive alternative sampling strategies, such as dried blood spot sampling or volumetric absorptive microsampling (VAMS). A VAMS-based multi-mycotoxin method was developed and validated for 24 different mycotoxins. Method validation was based on the Bioanalytical Method Validation Guideline of the Food and Drug Administration from the United States and for most of the studied mycotoxins, the results of the performance characteristics were in agreement with the criteria of the European Commission Decision 2002/657/EC. The recovery for the different mycotoxins was not haematocrit dependent and remained acceptable after storing the VAMS for 7 and 21 days at refrigeration temperature (4 °C) and room temperature, demonstrating that VAMS could be applied to assess mycotoxin exposure in blood in resource-limited areas, where there may be a delay between sampling and analysis. Finally, a comparison between VAMS and a procedure for liquid whole blood analysis, performed on 20 different blood samples, did not result in missed exposed cases for VAMS. Moreover, both methods detected similar levels of ochratoxin A, ochratoxin alpha, zearalenone and aflatoxin B1. Given all the benefits associated with VAMS and the developed method, VAMS sampling may serve as an alternative to conventional venous sampling to evaluate multiple mycotoxin exposure.

Analysis of Multiple Mycotoxins in the Qatari Population and Their Relation to Markers of Oxidative Stress.

Mycotoxins are naturally occurring food toxins worldwide that can cause serious health effects. The measurement of mycotoxin biomarkers in biological fluids is needed to assess individuals' exposure. The aim of this study was to investigate the incidence of mycotoxins in the Qatari population. Serum samples from 412 adults and urinary samples from 559 adults were analyzed for the presence of mycotoxin biomarkers. Multimycotoxin approaches have been applied, using liquid chromatography mass spectrometry methods. Samples were further analyzed for the oxidative stress markers and compared with regard to the incidence of mycotoxins. The presence of mycotoxins was identified in 37% of serum samples and in less than 20% of urine samples. It was found that 88% of positive of the samples were positive for only one mycotoxin, while 12% of positive samples had two or more mycotoxins. Trichothecenes and zearalenone metabolites were most commonly detected mycotoxins, followed by aflatoxins, roquefortine C and mycophenolic acid. The presence of mycotoxins was found to positively correlate with oxidative stress markers. The obtained results illustrate the importance of mycotoxin biomonitoring studies in humans and the need to elucidate the underlying mechanisms of mycotoxin-induced toxicity.

Assessment of Exposure to Mycotoxins in Spanish Children through the Analysis of Their Levels in Plasma Samples.

In this study, we present, for the first time in Spain, the levels of 19 mycotoxins in plasma samples from healthy and sick children (digestive, autism spectrum (ASD), and attention deficit hyperactivity (ADHD) disorders) (n = 79, aged 2-16). The samples were analyzed by liquid chromatography-mass spectrometry (triple quadrupole) (LC-MS/MS). To detect Phase II metabolites, the samples were reanalyzed after pre-treatment with ß-glucuronidase/arylsulfatase. The most prevalent mycotoxin was ochratoxin A (OTA) in all groups of children, before and after enzyme treatment. In healthy children, the incidence of OTA was 92.5% in both cases and higher than in sick children before (36.7% in digestive disorders, 50% in ASD, and 14.3% in ADHD) and also after the enzymatic treatment (76.6 % in digestive disorders, 50% in ASD, and 85.7% in ADHD). OTA levels increased in over 40% of healthy children after enzymatic treatment, and this increase in incidence and levels was also observed in all sick children. This suggests the presence of OTA conjugates in plasma. In addition, differences in OTA metabolism may be assumed. OTA levels are higher in healthy children, even after enzymatic treatment (mean OTA value for healthy children 3.29 ng/mL, 1.90 ng/mL for digestive disorders, 1.90 ng/mL for ASD, and 0.82 ng/mL for ADHD). Ochratoxin B appears only in the samples of healthy children with a low incidence (11.4%), always co-occurring with OTA. Sterigmatocystin (STER) was detected after enzymatic hydrolysis with a high incidence in all groups, especially in sick children (98.7% in healthy children and 100% in patients). This supports glucuronidation as a pathway for STER metabolism in children. Although other mycotoxins were studied (aflatoxins B1, B2, G1, G2, and M1; T-2 and HT-2 toxins; deoxynivalenol, deepoxy-deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol; zearalenone; nivalenol; fusarenon-X; neosolaniol; and diacetoxyscirpenol), they were not detected either before or after enzymatic treatment in any of the groups of children. In conclusion, OTA and STER should be highly considered in the risk assessment of mycotoxins. Studies concerning their sources of exposure, toxicokinetics, and the relationship between plasma levels and toxic effects are of utmost importance in children.

Improved methods for biomarker analysis of the big five mycotoxins enables reliable exposure characterization in a population of childbearing age women in Rwanda.

Of the five agriculturally important mycotoxins, AFB1, FB1, DON, ZEA and OTA, a well-characterized biomarker of exposure in blood is only available for aflatoxin. Working with a population of 139 women of childbearing age in Rwanda, we undertook a comprehensive assessment of their dietary mycotoxin exposure. Using high-resolution LC-MS/MS with stable isotope dilution analysis, the albumin-aflatoxin adduct was quantitated in plasma. Similarly, AFM1, AFB1, AFG1, FB1 and B2, OTA, zearalenone, α-zearalenol, deoxynivalenol, deoxynivalenol-15-glucuronide and deoxynivalenol-3-glucuronide were quantitated in urine. AFB1-Lys was detected in plasma from 81% of the women, indicative of exposures 1-2 orders of magnitude above current guidance. Zearalenone and/or α-zearalenol were detected in the urine of 61% of the women, the majority of whom had estimated exposures 2-5 times the PMTDI, with one third more than an order of magnitude above. Urinary deoxynivalenol or the two glucuronide conjugates were found in 77% of the participants. Of these, 60% were below the PMTDI, 28% were twice and 12% were >10x the PMTDI. Fumonisin B1 (30%) and ochratoxin A (71%) were also detected in urine. Exposures observed in these Rwandan women raise serious food safety concerns and highlight the need for authorities to help manage multiple mycotoxins in their diet.

Effects of the mycotoxin metabolite de-epoxy-deoxynivalenol (DOM-1) on embryo development and sperm motility in cattle.

Contamination of animal feed with Fusarium spp results in accumulation of mycotoxins including deoxynivalenol. In animals, deoxynivalenol is metabolized to de-epoxy deoxynivalenol (DOM-1), which is generally considered to be a non-toxic metabolite; however, recent studies demonstrated that DOM-1 can reduce steroid production and induce apoptosis in the bovine ovary. The objectives of this study were to assess the effects of DOM-1 on applied aspects of reproductive function in cattle, specifically sperm function and embryo development in vitro and follicle growth and superovulatory responses in vivo. The effect of naturally contaminated feed on superovulatory responses was assessed; a dose of 6 ppm deoxynivalenol increased blood DOM-1 concentrations to 20 ng/ml, but this did not alter the number of viable embryos recovered on day 7. However, intrafollicular injection of DOM-1 (100 ng/ml) directly into the growing dominant follicle resulted in cessation of follicular growth over the subsequent 3 days. Treatment with DOM-1 reduced motility of bull spermatozoa over a 10-h period in vitro. Addition of DOM-1 to oocytes in vitro during IVM did not alter rates of cumulus expansion and nuclear maturation, but treatment during IVF reduced the rate of blastocyst formation. These data illustrate that DOM-1 is more biologically active than previously thought and negatively impacted reproductive outcomes in cattle.

Aflatoxin B1 exposure and liver cirrhosis in Guatemala: a case-control study.

OBJECTIVE: In Guatemala, cirrhosis is among the 10 leading causes of death, and mortality rates have increased lately. The reasons for this heavy burden of disease are not clear as the prevalence of prominent risk factors, such as hepatitis B virus, hepatitis C virus and heavy alcohol consumption, appears to be low. Aflatoxin B1 (AFB1) exposure, however, appears to be high, and thus could be associated with the high burden of cirrhosis. Whether AFB1 increases the risk of cirrhosis in the absence of viral infection, however, is not clear. DESIGN: Cirrhosis cases (n=100) from two major referral hospitals in Guatemala City were compared with controls (n=200) from a cross-sectional study. Logistic regression was used to estimate the ORs and 95% CIs of cirrhosis and quintiles of AFB1 in crude and adjusted models. A sex-stratified analysis was also conducted. RESULTS: The median AFB1 level was significantly higher among the cases (11.4 pg/mg) than controls (5.11 pg/mg). In logistic regression analyses, higher levels of AFB1 was associated with cirrhosis (quintile 5 vs quintile 1, OR: 11.55; 95% CI 4.05 to 32.89). No attenuation was observed with adjustment by sex, ethnicity, hepatitis B virus status, and heavy alcohol consumption. A significantly increasing trend in association was observed in both models (p trend <0.01). Additionally, the cirrhosis-AFB1 association was more prominent among men. CONCLUSIONS: The current study found a significant positive association between AFB1 exposure and cirrhosis. Mitigation of AFB1 exposure and a better understanding of additional risk factors may be important to reduce the burden of cirrhosis in Guatemala.

In Vitro Toxicokinetics and Phase I Biotransformation of the Mycotoxin Penitrem A in Dogs.

The tremorgenic mycotoxin penitrem A is produced by Penicillium species as a secondary metabolite on moldy food and feed. Dogs are sometimes exposed to penitrem A by consumption of spoiled food waste or fallen fruit. The lipophilic toxin crosses the blood-brain barrier and targets neuroreceptors and neurotransmitter release mechanisms in the central and peripheral nervous systems. Typical symptoms of penitrem A intoxication are periodical or continuous tremors, which can be passing, persistent or lethal, depending on the absorbed dose. There is presently no information on the biotransformation and toxicokinetics of penitrem A in dogs. The aim of the present study was therefore to identify potential metabolites of the toxin by performing in vitro biotransformation assays in dog liver microsomes. Analyses by liquid chromatography coupled to high-resolution mass spectrometry led to the provisional identification of eleven penitrem A phase I metabolites, which were tentatively characterized as various oxidation products. Furthermore, elimination parameters determined in in vitro assays run under linear kinetics were used for in vitro-to-in vivo extrapolation of the toxicokinetic data, predicting a maximal bioavailability of more than 50%. The metabolite profile detected in the in vitro assays was similar to that observed in the plasma of an intoxicated dog, confirming the predictive capability of the in vitro approach.

Human Biomonitoring of Mycotoxins in Blood, Plasma and Serum in Recent Years: A Review.

This manuscript reviews the state-of-the-art regarding human biological monitoring (HBM) of mycotoxins in plasma serum and blood samples. After a comprehensive and systematic literature review, with a focus on the last five years, several aspects were analyzed and summarized: a) the biomarkers analyzed and their encountered levels, b) the analytical methodologies developed and c) the relationship between biomarker levels and some illnesses. In the literature reviewed, aflatoxin B1-lysine (AFB1-lys) and ochratoxin A (OTA) in plasma and serum were the most widely studied mycotoxin biomarkers for HBM. Regarding analytical methodologies, a clear increase in the development of methods for the simultaneous determination of multiple mycotoxins has been observed. For this purpose, the use of liquid chromatography (LC) methodologies, especially when coupled with tandem mass spectrometry (MS/MS) or high resolution mass spectrometry (HRMS), has grown. A high percentage of the samples analyzed for OTA or aflatoxin B1 (mostly as AFB1-lys) in the reviewed papers were positive, demonstrating human exposure to mycotoxins. This review confirms the importance of mycotoxin human biomonitoring and highlights the important challenges that should be faced, such as the inclusion of other mycotoxins in HBM programs, the need to increase knowledge of mycotoxin metabolism and toxicokinetics, and the need for reference materials and new methodologies for treating samples. In addition, guidelines are required for analytical method validation, as well as equations to establish the relationship between human fluid levels and mycotoxin intake.

Mycotoxins in blood and urine of Swedish adolescents-possible associations to food intake and other background characteristics.

The exposure to mycotoxins of Swedish adolescents is currently unknown. The aim of the present study was to investigate the exposure to mycotoxins and their association with food intake, and background characteristics in adolescents of a national dietary survey. About 3000 school students (1000 from the 5th, 8th and 11th school years) were recruited for the survey. The participants completed Web-based questionnaires on food propensity, sociodemography and health, and a Web-based dietary recall. Spot urine and blood samples were collected from 1105 of the participants for mycotoxin biomarker analysis. Mycotoxins were analysed with multibiomarker methods in urine (HPLC-MS/MS) and serum (HPLC-MS/MS). Of the 35 different analytes in urine, the frequency of positive samples were the following: deoxynivalenol (DON, 4.8%), DON-15-ß-D-O-glucuronide (DON-15GlcA, 9.1%), dihydro-citrinone (DH-CIT, 0.5%), HT-2-glucuronide (HT-2-3-GlcA, 0.1%) and ochratoxin A (OTA, 0.1%). Of the 27 different analytes in serum, OTA was detected in all samples, while 2'R-ochratoxin A (2'R-OTA) was found in 8.3% and enniatin B (EnB) in 99.2% of the samples. Exposure assessment calculations were performed on OTA from the serum concentration and on DON equivalents (DON eqv) from the urine concentration. All probable daily intake (PDI) estimates were below tolerable daily intakes, except for 1.6% of the participants for DON. The maximum PDI was 4.3 µg DON eqv/kg body weight and day. Consumption of cereal grain commodities was associated with levels of DON, EnB or OTA in biofluids. Serum OTA was also associated with intakes of raisins and coffee. Furthermore, coffee consumption correlated well with 2'R-OTA concentration in serum. In conclusion, exposure to mycotoxins in Swedish adolescents is common, but fortunately, high exposure was rare.

Development and validation of a methodology based on Captiva EMR-lipid clean-up and LC-MS/MS analysis for the simultaneous determination of mycotoxins in human plasma.

We report the methodology for the quantification of 19 mycotoxins in human plasma using high performance liquid chromatography-mass spectrometry (triple quadrupole). The studied mycotoxins were: deepoxy-deoxynivalenol, aflatoxins (B1, B2, G1, G2 and M1), T-2 and HT-2, ochratoxins A and B, zearalenone, sterigmatocystin, nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, diacetoxyscirpenol and fusarenon-X. Sample deproteinization and cleanup were performed in one step using Captiva EMR-lipid (3 mL) cartridges and acetonitrile (with 1% formic acid). The extraction step was simple and fast. Validation was based on the evaluation of limits of detection (LOD) and quantification, linearity, precision, recovery, matrix effect, and stability. LOD values ranged from 0.04 ng/mL for aflatoxin B1 to 2.7 ng/mL for HT-2, except for nivalenol, which was 9.1 ng/mL. Recovery was obtained in intermediate precision conditions and at three concentration levels. Mean values ranged from 68.8% for sterigmatocystin to 97.6% for diacetoxyscirpenol (RDS ≤ 15% for all the mycotoxins). Matrix effects (assessed at three concentration levels and in intermediate conditions) were not significant for most of the mycotoxins and were between 75.4% for sterigmatocystin and 109.3% for ochratoxin B (RDS ≤ 15% for all the mycotoxins). This methodology will be useful in human biomonitoring studies of mycotoxins for its reliability.

Bioavailability, metabolism, and excretion of a complex Alternaria culture extract versus altertoxin II: a comparative study in rats.

Despite the frequent infection of agricultural crops by Alternaria spp., their toxic secondary metabolites and potential food contaminants lack comprehensive metabolic characterization. In this study, we investigated their bioavailability, metabolism, and excretion in vivo. A complex Alternaria culture extract (50 mg/kg body weight) containing 11 known toxins and the isolated lead toxin altertoxin II (0.7 mg/kg body weight) were administered per gavage to groups of 14 Sprague Dawley rats each. After 3 h and 24 h, plasma, urine and feces were collected to determine toxin recoveries. For reliable quantitation, an LC-MS/MS method for the simultaneous detection of 20 Alternaria toxins and metabolites was developed and optimized for either biological matrix. The obtained results demonstrated efficient excretion of alternariol (AOH) and its monomethyl ether (AME) via feces (> 89%) and urine (> 2.6%) after 24 h, while the majority of tenuazonic acid was recovered in urine (20 and 87% after 3 and 24 h, respectively). Moreover, modified forms of AOH and AME were identified in urine and fecal samples confirming both, mammalian phase-I (4-hydroxy-AOH) and phase-II (sulfates) biotransformation in vivo. Despite the comparably high doses, perylene quinones were recovered only at very low levels (altertoxin I, alterperylenol, < 0.06% in urine and plasma, < 5% in feces) or not at all (highly genotoxic, epoxide-holding altertoxin II, stemphyltoxin III). Interestingly, altertoxin I was detected in all matrices of rats receiving altertoxin II and suggests enzymatic de-epoxidation in vivo. In conclusion, the present study contributes valuable information to advance our understanding of the emerging Alternaria mycotoxins and their relevance on food safety.

Assessment of Dried Blood Spots for Multi-Mycotoxin Biomarker Analysis in Pigs and Broiler Chickens.

Dried blood spots (DBSs), a micro-sampling technique whereby a drop of blood is collected on filter paper has multiple advantages over conventional blood sampling regarding the sampling itself, as well as transportation and storage. This is the first paper describing the development and validation of a method for the determination of 23 mycotoxins and phase I metabolites in DBSs from pigs and broiler chickens using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The targeted mycotoxins belong to groups for which the occurrence in feed is regulated by the European Union, namely, aflatoxins, ochratoxin A and several Fusarium mycotoxins, and to two groups of unregulated mycotoxins, namely Alternaria mycotoxins and Fusarium mycotoxins (enniatins and beauvericin). The impact of blood haematocrit, DBS sampling volume and size of the analysed DBS disk on the validation results was assessed. No effects of variation in size of the analysed disk, haematocrit and spotted blood volume were observed for most mycotoxins, except for the aflatoxins and ß-zearalanol (BZAL) at the lowest haematocrit (26%) level and for the enniatins (ENNs) at the lowest volume (40 µL). The developed method was transferred to an LC-high resolution mass spectrometry instrument to determine phase II metabolites. Then, the DBS technique was applied in a proof-of-concept toxicokinetic study including a comparison with LC-MS/MS data from plasma obtained with conventional venous blood sampling. A strong correlation (r > 0.947) was observed between plasma and DBS concentrations. Finally, DBSs were also applied in a pilot exposure assessment study to test their applicability under field conditions.

The In Vivo and In Vitro Toxicokinetics of Citreoviridin Extracted from Penicillium citreonigrum.

Citreoviridin (CTVD), a mycotoxin called yellow rice toxin, is reported to be related to acute cardiac beriberi; however, its toxicokinetics remain unclear. The present study elucidated the toxicokinetics through in vivo experiments in swine and predicted the human toxicokinetics by comparing the findings to those from in vitro experiments. In vivo experiments revealed the high bioavailability of CTVD (116.4%) in swine. An intestinal permeability study using Caco-2 cells to estimate the toxicokinetics in humans showed that CTVD has a high permeability coefficient. When CTVD was incubated with hepatic S9 fraction from swine and humans, hydroxylation and methylation, desaturation, and dihydroxylation derivatives were produced as the predominant metabolites. The levels of these products produced using human S9 were higher than those obtained swine S9, while CTVD glucuronide was produced slowly in human S9 in comparison to swine S9. Furthermore, the elimination of CTVD by human S9 was significantly more rapid in comparison to that by swine S9. These results suggest that CTVD is easily absorbed in swine and that it remains in the body where it is slowly metabolized. In contrast, the absorption of CTVD in humans would be the same as that in swine, although its elimination would be faster.

Analyses of biomarkers of exposure to nephrotoxic mycotoxins in a cohort of patients with renal tumours.

The Czech Republic occupies the first place in the world in the frequency of renal and other urinary tract tumours, but their aetiology is unknown. To explore whether carcinogenic and nephrotoxic mycotoxins may contribute to kidney diseases in the Czech population, biomarkers of ochratoxin A (OTA) and citrinin (CIT) exposure were determined in biological specimens from a cohort of 50 patients with malignant renal tumours. Biomarker analyses in blood and urine samples used validated targeted methods for measuring OTA and CIT plus dihydrocitrinone (DH-CIT) after enrichment of analytes by specific immunoaffinity clean-up. OTA and CIT plus its metabolite DH-CIT were frequently detected in patient urine samples (OTA 62%; CIT 91%; DH-CIT 100%). The concentration ranges in urine were 1-27.8 ng/L for OTA, 2-87 ng/L for CIT and 2-160 ng/L for DH-CIT. The analyses of blood samples revealed also a frequent co-occurrence of OTA and CIT, in the ranges of 40-870 ng/L serum for OTA and 21-182 ng/L plasma for CIT. This first analysis of biomarkers in blood and urine samples of Czech patients revealed no major differences in comparison with published data for the general healthy Czech and European populations. Nonetheless, a frequent co-occurrence of CIT and OTA biomarkers in patient samples may be of interest with regard to potential interactions with other risk factors for renal disease.