Role of albumin in the metabolism and excretion of ochratoxin A.

Ochratoxin A (OTA) is known to be strongly bound to serum albumin, but it remains unknown how albumin affects its metabolism and kinetics. To close this gap, we used a mouse model, where heterozygous albumin deletion reduces serum albumin to concentrations similar to hypoalbuminemic patients and completely eliminates albumin by a homozygous knockout. OTA and its potential metabolites (OTα, 4-OH-OTA, 7'-OH-OTA, OTHQ, OP-OTA, OTB-GSH, OTB-NAC, OTB) were time-dependently analyzed in plasma, bile, and urine by LC-MS/MS and were compared to previously published hepatotoxicity and nephrotoxicity data. Homozygous albumin deletion strongly accelerated plasma clearance as well as biliary and urinary excretion of the parent compound and its hydroxylation products. Decreasing albumin in mice by the heterozygous and even more by the homozygous knockout leads to an increase in the parent compound in urine which corresponded to increased nephrotoxicity. The role of albumin in OTA-induced hepatotoxicity is more complex, since heterozygous but not homozygous nor wild-type mice showed a strong biliary increase in the toxic open lactone OP-OTA. Correspondingly, OTA-induced hepatotoxicity was higher in heterozygous than in wild-type and homozygous animals. We present evidence that albumin-mediated retention of OTA in hepatocytes is required for formation of the toxic OP-OTA, while complete albumin elimination leads to rapid biliary clearance of OTA from hepatocytes with less formation of OP-OTA. In conclusion, albumin has a strong influence on metabolism and toxicity of OTA. In hypoalbuminemia, the parent OTA is associated with increased nephrotoxicity and the open lactone with increased hepatotoxicity.

Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Aflatoxin M1 Analysis in Urine of Mill Workers in Bangladesh: A Pilot Study.

Presence of aflatoxin B1 (AFB1) in food and feed is a serious problem, especially in developing countries. Human exposure to this carcinogenic mycotoxin can occur through dietary intake, but also through inhalation or dermal contact when handling and processing AFB1-contaminated crops. A suitable biomarker of AFB1 exposure by all routes is the occurrence of its hydroxylated metabolite aflatoxin M1 (AFM1) in urine. To assess mycotoxin exposure in mill workers in Bangladesh, we analyzed AFM1 levels in urine samples of this population group who may encounter both dietary and occupational AFB1 exposure. In this pilot study, a total of 76 participants (51 mill workers and 25 controls) were enrolled from the Sylhet region of Bangladesh. Urine samples were collected from people who worked in rice, wheat, maize and spice mills and from controls with no occupational contact to these materials. A questionnaire was used to collect information on basic characteristics and normal food habits of all participants. Levels of AFM1 in the urine samples were determined by a competitive enzyme linked immunosorbent assay. AFM1 was detected in 96.1% of mill workers' urine samples with a range of LOD (40) of 217.7 pg/mL and also in 92% of control subject's urine samples with a range of LOD of 307.0 pg/mL). The mean level of AFM1 in mill workers' urine (106.5 ± 35.0 pg/mL) was slightly lower than that of the control group (123.3 ± 52.4 pg/mL), whilst the mean AFM1 urinary level adjusted for creatinine was higher in mill workers (142.1 ± 126.1 pg/mg crea) than in the control group (98.5 ± 71.2 pg/mg crea). Yet, these differences in biomarker levels were not statistically significant. Slightly different mean urinary AFM1 levels were observed between maize mill, spice mill, rice mill, and wheat mill workers, yet biomarker values are based on a small number of individuals in these subgroups. No significant correlations were found between the study subjects' urine AFM1 levels and their consumption of some staple food items, except for a significant correlation observed between urinary biomarker levels and consumption of groundnuts. In conclusion, this pilot study revealed the frequent presence of AFM1 in the urine of mill workers in Bangladesh and those of concurrent controls with dietary AFB1 exposure only. The absence of a statistical difference in mean biomarker levels for workers and controls suggests that in the specific setting, no extra occupational exposure occurred. Yet, the high prevalence of non-negligible AFM1 levels in the collected urines encourage further studies in Bangladesh regarding aflatoxin exposure.

Occurrence of ochratoxin A in breast milk and urine samples of nursing mothers in Bangladesh.

The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin with carcinogenic properties and, thus, of concern as a food contaminant. Since food contaminant data are scarce in Bangladesh, we applied human biomonitoring to gain more insights into OTA exposure in the country's population. OTA concentrations in human milk and urine samples of nursing mothers were determined with the aim to assess also exposure to this mycotoxin in breastfed infants. Breastfeeding mothers (n = 74) from three districts of Bangladesh (Sylhet, Cumilla, and Mymensingh region) participated in this study. They provided demographic data, along with breast milk and urine samples. OTA levels were measured by a competitive enzyme-linked immunosorbent assay (ELISA) with a detection limit of 60 ng/L for milk and 30 ng/L for urine.OTA was detected in 62.2% of all breast milk samples (mean 74.8 ± 49.0 ng/L, range < LOD-243.3 ng/L) and in 51.4% of all urine samples (mean 44.3 ± 63.5 ng/L, range < LOD-519.3 ng/L). The differences observed between regions for mean breast milk or for urinary OTA levels were relatively small. No significant correlation was observed between OTA levels in breast milk and food consumption patterns among nursing mothers. Regarding infant exposure, the estimated average daily intake of OTA for all was 15.0 ng/kg bw/day (range 4.5-45 ng/kg bw/day). In 34.5% of these infants, their estimated daily OTA intake exceeded a preliminary TDI value set by EFSA (17 ng/kg bw/day). The mean OTA intake was slightly higher (16.2 ± 7.8 ng/kg bw/day) in 1-2 months babies than in older infants (< 2 to 12 months), although the difference was not significant. Presence of OTA in most milk and urine samples of nursing mothers documents their widespread dietary mycotoxin exposure. Although based on a relatively small number of participants, the present analysis indicates non-negligible exposure of some nursed infants in Bangladesh. Therefore, further biomonitoring studies and investigations on major sources of OTA in food commodities are encouraged.

Comparative metabolism of aflatoxin B1 in mouse, rat and human primary hepatocytes using HPLC-MS/MS.

Aflatoxin B1 (AFB1) is a highly hepatotoxic and carcinogenic mycotoxin produced by Aspergillus species. The compound is mainly metabolized in the liver and its metabolism varies between species. The present study quantified relevant AFB1- metabolites formed by mouse, rat, and human primary hepatocytes after treatment with 1 µM and 10 µM AFB1. The use of liquid chromatographic separation coupled with tandem mass spectrometric detection enabled the selective and sensitive determination of phase I and phase II metabolites of AFB1 over incubation times of up to 24 h. The binding of AFB1 to macromolecules was also considered. The fastest metabolism of AFB1 was observed in mouse hepatocytes which formed aflatoxin P1 as a major metabolite and also its glucuronidated form, while AFP1 occurred only in traces in the other species. Aflatoxin M1 was formed in all species and was, together with aflatoxin Q1 and aflatoxicol, the main metabolite in human cells. Effective epoxidation led to high amounts of DNA adducts already 30 min post-treatment, especially in rat hepatocytes. Lower levels of DNA adducts and fast DNA repair were found in mouse hepatocytes. Also, protein adducts arising from reactive intermediates were formed rapidly in all three species. Detoxification via glutathione conjugation and subsequent formation of the N-acetylcysteine derivative appeared to be similar in mice and in rats and strongly differed from human hepatocytes which did not form these metabolites at all. The use of qualitative reference material of a multitude of metabolites and the comparison of hepatocyte metabolism in three species using advanced methods enabled considerations on toxification and detoxification mechanisms of AFB1. In addition to glutathione conjugation, phase I metabolism is strongly involved in the detoxification of AFB1.

Correction: Degen et al. Citrinin Exposure in Germany: Urine Biomarker Analysis in Children and Adults. Toxins 2023, 15, 26.

In the original publication [...].

Inhibition of cytochrome P450 enhances the nephro- and hepatotoxicity of ochratoxin A.

The mycotoxin ochratoxin A (OTA) is a contaminant in food that causes nephrotoxicity and to a minor degree hepatotoxicity. Recently, we observed that OTA induces liver damage preferentially to the cytochrome P450 (CYP)-expressing pericentral lobular zone, similar to hepatotoxic substances known to be metabolically toxified by CYP, such as acetaminophen or carbon tetrachloride. To investigate whether CYP influences OTA toxicity, we used a single dose of OTA (7.5 mg/kg; intravenous) with and without pre-treatment with the pan CYP-inhibitor 1-aminobenzotriazole (ABT) 2 h before OTA administration. Blood, urine, as well as liver and kidney tissue samples were collected 24 h after OTA administration for biochemical and histopathological analyses. Inhibition of CYPs by ABT strongly increased the nephro- and hepatotoxicity of OTA. The urinary kidney damage biomarkers kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) were increased > 126-fold and > 20-fold, respectively, in mice treated with ABT and OTA compared to those receiving OTA alone. The blood biomarkers of liver damage, alanine transaminase (ALT) and aspartate transaminase (AST) both increased > 21- and 30-fold, respectively, when OTA was administered to ABT pre-treated mice compared to the effect of OTA alone. Histological analysis of the liver revealed a pericentral lobular damage induced by OTA despite CYP-inhibition by ABT. Administration of ABT alone caused no hepato- or nephrotoxicity. Overall, the results presented are compatible with a scenario where CYPs mediate the detoxification of OTA, yet the mechanisms responsible for the pericental liver damage pattern still remain to be elucidated.

Hypoalbuminemia affects the spatio-temporal tissue distribution of ochratoxin A in liver and kidneys: consequences for organ toxicity.

Hypoalbuminemia (HA) is frequently observed in systemic inflammatory diseases and in liver disease. However, the influence of HA on the pharmacokinetics and toxicity of compounds with high plasma albumin binding remained insufficiently studied. The 'lack-of-delivery-concept' postulates that HA leads to less carrier mediated uptake of albumin bound substances into hepatocytes and to less glomerular filtration; in contrast, the 'concept-of-higher-free-fraction' argues that increased concentrations of non-albumin bound compounds facilitate hepatocellular uptake and enhance glomerular filtration. To address this question, we performed intravital imaging on livers and kidneys of anesthetized mice to quantify the spatio-temporal tissue distribution of the mycotoxin ochratoxin A (OTA) based on its auto-fluorescence in albumin knockout and wild-type mice. HA strongly enhanced the uptake of OTA from the sinusoidal blood into hepatocytes, followed by faster secretion into bile canaliculi. These toxicokinetic changes were associated with increased hepatotoxicity in heterozygous albumin knockout mice for which serum albumin was reduced to a similar extent as in patients with severe hypoalbuminemia. HA also led to a shorter half-life of OTA in renal capillaries, increased glomerular filtration, and to enhanced uptake of OTA into tubular epithelial cells. In conclusion, the results favor the 'concept-of-higher-free-fraction' in HA; accordingly, HA causes an increased tissue uptake of compounds with high albumin binding and increased organ toxicity. It should be studied if this concept can be generalized to all compounds with high plasma albumin binding that are substrates of hepatocyte and renal tubular epithelial cell carriers.

Assessment of multiple mycotoxin exposure and its association with food consumption: a human biomonitoring study in a pregnant cohort in rural Bangladesh.

Aflatoxins (AFs), ochratoxin A (OTA), citrinin (CIT), fumonisin B1 (FB1), zearalenone (ZEN), and deoxynivalenol (DON) are mycotoxins that may contaminate diets, especially in low-income settings, with potentially severe health consequences. This study investigates the exposure of 439 pregnant women in rural Bangladesh to 35 mycotoxins and their corresponding health risks and links their exposure to certain foods and local stimulants. Overall, 447 first-morning urine samples were collected from pregnant women between July 2018 and November 2019. Mycotoxin biomarkers were quantified by DaS-HPLC-MS/MS. Urinary concentration of frequently occurring mycotoxins was used to estimate dietary mycotoxin exposure. Median regression analyses were performed to investigate the association between the consumption of certain foods and local stimulants, and urinary concentration of frequently occurring mycotoxins. Only in 17 of 447 urine samples (4%) were none of the investigated mycotoxins detected. Biomarkers for six major mycotoxins (AFs, CIT, DON, FB1, OTA, and ZEN) were detected in the urine samples. OTA (95%), CIT (61%), and DON (6%) were most frequently detected, with multiple mycotoxins co-occurring in 281/447 (63%) of urine samples. Under the lowest exposure scenario, dietary exposure to OTA, CIT, and DON was of public health concern in 95%, 16%, and 1% of the pregnant women, respectively. Consumption of specific foods and local stimulants-betel nut, betel leaf, and chewing tobacco-were associated with OTA, CIT, and DON urine levels. In conclusion, exposure to multiple mycotoxins during early pregnancy is widespread in this rural community and represents a potential health risk for mothers and their offspring.

Citrinin Exposure in Germany: Urine Biomarker Analysis in Children and Adults.

Citrinin (CIT), a mycotoxin known to exert nephrotoxicity, is a contaminant in food and feed. Since CIT contamination is not regularly analyzed, data on its occurrence and especially levels in food commodities are insufficient for conducting a conventional exposure assessment. Yet, human biomonitoring, i.e., an analysis of CIT and its metabolite dihydrocitrinone (DH-CIT) in urine samples allows to estimate exposure. This study investigated CIT exposure in young (2-14 years) and adult (24-61 years) residents of three federal states in Germany. A total of 179 urine samples from children and 142 from adults were collected and analyzed by a targeted LC-MS/MS based method for presence of CIT and DH-CIT. At least one of the biomarkers was detected and quantified in all urines, which indicated a widespread dietary exposure to the mycotoxin in Germany. Interestingly, the biomarker concentrations of CITtotal (sum of CIT and DH-CIT) were higher in children's urine (range 0.05-7.62 ng/mL; median of 0.54 ng/mL) than in urines from adults (range 0.04-3.5 ng/mL; median 0.3 ng/mL). The biomarker levels (CITtotal) of individual urines served to calculate the probable daily CIT intake, for comparison to a value of 0.2 µg/kg bw/day defined as 'level of no concern for nephrotoxicity' by the European Food Safety Authority. The median exposure of German adults was 0.013 µg/kg b.w., with only one urine donor exceeding this provisional tolerable daily intake (pTDI) for CIT. The median exposure of children was 0.05 µg/kg bw per day (i.e., 25% of the pTDI); however, CIT exposure in 12 individuals (6.3% of our study group) exceeded the limit value, with a maximum intake of 0.46 µg/kg b.w. per day. In conclusion, these results show evidence for non-negligible exposure to CIT in some individuals in Germany, mainly in children. Therefore, further biomonitoring studies and investigations aimed to identify the major sources of CIT exposure in food commodities are required.

The Presence of Aflatoxin M1 in Milk and Milk Products in Bangladesh.

As milk provides both micro- and macronutrients, it is an important component in the diet. However, the presence of aflatoxin B1 (AFB1) in the feed of dairy cattle results in contamination of milk and dairy products with aflatoxin M1 (AFM1), a toxic metabolite of the carcinogenic mycotoxin. With the aim to determine AFM1 concentrations in milk and milk products consumed in Bangladesh, in total, 145 samples were collected in four divisional regions (Sylhet, Dhaka, Chittagong, and Rajshahi). The samples comprised these categories: raw milk (n = 105), pasteurized milk (n = 15), ultra-high temperature (UHT)-treated milk (n = 15), fermented milk products such as yogurt (n = 5), and milk powder (n = 5). AFM1 levels in these samples were determined through competitive enzyme-linked immunosorbent assay (ELISA). Overall, AFM1 was present in 78.6% of milk and milk products in the range of 5.0 to 198.7 ng/L. AFM1 was detected in 71.4% of raw milk (mean 41.1, range 5.0-198.7 ng/L), and in all pasteurized milk (mean 106, range 17.2-187.7 ng/L) and UHT milk (mean 73, range 12.2-146.9 ng/L) samples. Lower AFM1 levels were found in yogurt (mean 16.9, range 8.3-41.1 ng/L) and milk powder samples (mean 6.6, range 5.9-7.0 ng/L). About one-third of the raw, pasteurized, and UHT milk samples exceeded the EU regulatory limit (50 ng/L) for AFM1 in milk, while AFM1 levels in yogurt and milk powder samples were well below this limit. Regarding regions, lower AFM1 contamination was observed in Chittagong (mean 6.6, max 10.6 ng/L), compared to Sylhet (mean 53.7, max 198.7 ng/L), Dhaka (mean 37.8, max 97.2 ng/L), and Rajshahi (mean 34.8, max 131.4 ng/L). Yet, no significant difference was observed in AFM1 levels between summer and winter season. In conclusion, the observed frequency and levels of aflatoxin contamination raise concern and must encourage further monitoring of AFM1 in milk and milk products in Bangladesh.

Occurrence of aflatoxin M1 in human breast milk in Bangladesh.

Breast milk is the best, most complete form of nutrition for newborns and infants. However, human milk can contain aflatoxin M1 (AFM1) upon ingestion of dietary mycotoxin contaminants, namely, aflatoxin B1 (AFB1), by lactating mothers. AFB1 and its hydroxylated metabolite AFM1 are potent carcinogens and thus an important issue in food safety and public health. This study is the first to explore the presence of AFM1 in breast milk samples from Bangladesh and assess infant exposure to this toxin, as a consequence of maternal mycotoxin intake. A total of 62 breast milk samples were collected from nursing mothers in Sylhet region of Bangladesh. The milk samples were collected between October 2019 and March 2020 and analyzed by a sensitive enzyme-linked immunosorbent assay. AFM1 was detected in 51.6% of the breast milk samples (colostrum, transitional and mature milk), with a mean concentration of 4.42 ± 0.56 pg/mL, and in the range between LOD (4.0 pg/mL) and 6.66 pg/mL. The frequent detection of AFM1 in breast milk indicates widespread dietary exposure to mycotoxins in our cohort. The estimated average daily intake of AFM1 for all nursed infants was 0.49 ng/kg b.w./day. No significant correlations were observed between AFM1 levels in human milk and food items regularly consumed by nursing women. Overall, AFM1 levels in breast milk samples from the Sylhet region of Bangladesh are moderate, and lower than the permissible levels established for AFM1 in dairy milk or infant formulae (50 and 25 ng/kg, respectively). Yet, this first data for AFM1 breast milk contaminant levels just reflect the recent situation in one cohort, and monitoring should be continued.

Subcellular spatio-temporal intravital kinetics of aflatoxin B1 and ochratoxin A in liver and kidney.

Local accumulation of xenobiotics in human and animal tissues may cause adverse effects. Large differences in their concentrations may exist between individual cell types, often due to the expression of specific uptake and export carriers. Here we established a two-photon microscopy-based technique for spatio-temporal detection of the distribution of mycotoxins in intact kidneys and livers of anesthetized mice with subcellular resolution. The mycotoxins ochratoxin A (OTA, 10 mg/kg b.w.) and aflatoxin B1 (AFB1, 1.5 mg/kg b.w.), which both show blue auto-fluorescence, were analyzed after intravenous bolus injections. Within seconds after administration, OTA was filtered by glomeruli, and enriched in distal tubular epithelial cells (dTEC). A striking feature of AFB1 toxicokinetics was its very rapid uptake from sinusoidal blood into hepatocytes (t1/2 ~ 4 min) and excretion into bile canaliculi. Interestingly, AFB1 was enriched in the nuclei of hepatocytes with zonal differences in clearance. In the cytoplasm of pericentral hepatocytes, the half-life (t1/2~ 63 min) was much longer compared to periportal hepatocytes of the same lobules (t1/2 ~ 9 min). In addition, nuclear AFB1 from periportal hepatocytes cleared faster compared to the pericentral region. These local differences in AFB1 clearance may be due to the pericentral expression of cytochrome P450 enzymes that activate AFB1 to protein- and DNA-binding metabolites. In conclusion, the present study shows that large spatio-temporal concentration differences exist within the same tissues and its analysis may provide valuable additional information to conventional toxicokinetic studies.

Determination of aflatoxin M1 and deoxynivalenol biomarkers in infants and children urines from Bangladesh.

The mycotoxins aflatoxin B1 (AFB1) and deoxynivalenol (DON) are found worldwide in crops and dietary staples. The prevalence and levels of these contaminants can vary greatly, and data in Bangladeshi food commodities are scarce. To characterize human exposure, we have conducted biomonitoring, analyzing AFM1 (a metabolite of AFB1) and DON levels in urines of adult cohorts in Bangladesh. Yet, AFM1 and DON occurrence has not been studied in the very young population of this country. Thus, the same methods, HPLC-FD for AFM1 and LC-MS/MS for DON analysis, were now applied to determine these biomarkers in urines of infants (n = 49) and young children (n = 105) in Rajshahi and Dhaka district. Overall, AFM1 and DON detection frequency was 43.5% and 33.4%, with 34.7% and 11.5% in infant and 47.6% and 39.4% in children urines, respectively. The mean AFM1 levels in all infants (9.1 ± 14.3, max 55.6 pg/mL) and children (8.8 ± 12.9, max 75.3 pg/mL) were not significantly different. The AFM1 mean level was slightly higher in Dhaka (9.4 ± 12.4) compared to Rajshahi (8.5 ± 13.9 pg/mL) district. The average DON level was about 2-fold higher in infant (3.8 ± 2.9, max 6.8 ng/mL) than children urines (1.6 ± 1.8, max 8.6 ng/mL), and higher in Rajshahi (2.1 ± 2.3 ng/mL) than Dhaka (1.4 ± 1.6 ng/mL) district. The biomarker-based estimated average daily DON intake (29.6 ± 108.3 ng/kg bw in infants and 36.4 ± 81.8 ng/kg bw in children) or the maximum exposure (560 ng/kg bw) do not exceed the current maximum provisional tolerable daily intake value of 1 µg/kg bw for DON, although DON exposure in infants and children is higher than that of Bangladeshi adults. The AFM1 urine levels in young children are somewhat lower than those found previously in adult cohorts in Bangladesh, but the frequent detection of this biomarker for AFB1 exposure raises further concerns, also for this vulnerable part of the population. Therefore, continuous surveillance for aflatoxins in Bangladeshi food commodities is clearly required, first to identify major sources of intake and then to reduce exposure.

Biological monitoring for ochratoxin A and citrinin and their metabolites in urine samples of infants and children in Bangladesh.

Ochratoxin A (OTA) and citrinin (CIT) are nephrotoxic mycotoxins, found in various foodstuffs and in animal feed, and may cause adverse effects on animal and human health. Previous biomonitoring data indicate a frequent co-exposure of Bangladeshi adults to these mycotoxins. However, since such data are not yet available for young children, a vulnerable part of the population, we conducted this study to assess their exposure to OTA and CIT and compare it with that of adults in Bangladesh. In total, 154 urine samples were collected from infants and children in Rajshahi (n = 88) and Dhaka (n = 66) district of Bangladesh. OTA, CIT, and their metabolites were analyzed by a sensitive HPLC-FLD or LC-MS/MS method, respectively. Overall, OTA and CIT biomarkers were detectable in 72.7% and 54.9% of urines, respectively. The mean OTA and OTα levels in urines were higher in children (0.13 ng/mL and 0.28 ng/mL, respectively) than in infants (0.08 ng/mL and 0.05 ng/mL, respectively). Regarding region, the mean level of OTA was higher in samples from Rajshahi district (0.13 ng/mL) than from Dhaka district (0.09 ng/mL), while the mean OTα level was 2-fold higher in the Dhaka. The total CIT biomarker concentration was significantly higher in children (2.16 ng/mL) than in infant (0.70 ng/mL) urines (p < 0.05), and the mean concentration of HO-CIT was about 6-fold higher than that of parent compound CIT. A provisional daily intake for CIT was calculated and exceeded a preliminary value set by EFSA (0.2 µg/kg bw) in 23.3% and 11.9% of children and infants, respectively. OTA and CIT biomarker concentrations in the young children cohorts are higher than those found in Bangladeshi adults in summer, but lower than in winter season. The new results indicate frequent co-exposure to nephrotoxic mycotoxins that varies between the cohorts and regions in Bangladesh.

Citrinin biomarkers: a review of recent data and application to human exposure assessment.

The mycotoxin citrinin (CIT) deserves attention due to its known toxic effects in mammalian species and a widespread occurrence in food commodities, often along with ochratoxin A, another nephrotoxic mycotoxin. Human exposure, a key element in assessing risks related to these food contaminants, depends upon mycotoxin levels in food and on food consumption. Yet, data available for CIT levels in food are insufficient for reliable intake estimates. Now biomonitoring, i.e., analysis of parent compound and/or metabolites in human specimen (blood, urine, breast milk), is increasingly used to investigate mycotoxin exposure. Biomonitoring requires sensitive methods for determining biomarkers of exposure, combined with kinetic data to conclude on the absorbed internal dose in an individual. Recent advances in LC-MS/MS-based analytical techniques have facilitated biomonitoring studies on the occurrence of CIT biomarkers in body fluids, mainly in urine samples. This review compiles evidence on human exposure to CIT in different countries, on CIT kinetics in humans, and on biomarker-based CIT intake estimates. Human CIT exposures are discussed in light of an intake value defined as 'level of no concern for nephrotoxicity' by the European Food Safety Agency, and some uncertainties in the toxicological data base. Further studies on CIT, including biomarker-based studies are warranted along with regular food surveys for this mycotoxin to protect consumers against undesirable health effects.

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.

Biomonitoring of zearalenone and its main metabolites in urines of Bangladeshi adults.

The Fusarium toxin zearalenone (ZEN) is of concern due to its pronounced estrogenic effects in mammalian species. ZEN contaminates various cereal crops and grain-based food along with modified forms which contribute to overall mycoestrogen exposure. As no data exist on the occurrence of ZEN in food commodities consumed in Bangladesh, we have analyzed ZEN and its main metabolites α-and ß-zearalenol (α-ZEL, ß-ZEL) by targeted LC-MS/MS method as biomarkers of exposure in urines (n = 62) from rural and urban residents in Rajshahi district collected in two seasons and from a pregnant women cohort (n = 20) in Dhaka district. Average levels of α-ZEL, the far more potent estrogenic metabolite, were clearly higher than those of ZEN and ß-ZEL. Biomarker levels in urban and rural residents showed some seasonal fluctuation: In winter urines, ZEN mean level was 0.040 ±â€¯0.037, α-ZEL 0.182 ±â€¯0.047 and ß-ZEL 0.018 ±â€¯0.016 ng/mL; in summer urines, ZEN mean was 0.028 ±â€¯0.015, α-ZEL 0.198 ±â€¯0.025 and ß-ZEL 0.013 ±â€¯0.005 ng/mL. In pregnant women, mean levels were: ZEN 0.057 ±â€¯0.041, α-ZEL 0.151 ±â€¯0.026 and ß-ZEL 0.055 ±â€¯0.057 ng/mL, thus similar to levels found in the Rajshahi cohort in winter season. Estimates of probable dietary mycoestrogen intake in the Bangladeshi adults reveal an exposure below the tolerable daily intake of 0.25 µg/kg b.w. set by EFSA.

Monitoring Early Life Mycotoxin Exposures via LC-MS/MS Breast Milk Analysis.

Infants are particularly susceptible toward the toxic effects of food contaminants, including mycotoxins. However, multimycotoxin exposure assessment in breast milk has received very limited attention so far, resulting in a poor understanding of coexposures during early life. Here, we present the development and application of a highly sensitive, specific, and quantitative assay assessing up to 28 mycotoxins, including regulated (aflatoxins, ochratoxin A, deoxynivalenol, zearalenone) and emerging mycotoxins as well as key metabolites by LC-MS/MS. After careful optimization of the sample preparation procedure, a QuEChERS protocol combined with a freeze-out step was validated in-house. The limits of quantification varied between 0.009 and 2.9 ng/mL, and for most analytes extraction recovery (74-116%) and intermediate precision (2-20%) were satisfactory. The method was applied to examine multiple breast milk samples obtained from 22 women ( n = 75 in total) from Ogun State, Nigeria. Most samples were either entirely free of mycotoxins or contaminated to a minimal extent with beauvericin (56%), enniatin B (9%), ochratoxin A (15%), and aflatoxin M1 (1%). The most abundant mycotoxin was beauvericin, which was not reported in this biological fluid before, with concentrations up to 0.019 ng/mL. In conclusion, the method demonstrated to be fit for purpose to determine and quantify low background contaminations in human breast milk. On the basis of the high sensitivity of the novel analytical method, it was possible to deduce that tolerable daily intake values were not exceeded by breastfeeding in the examined infants.