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.

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.

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.

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.

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.

Contribution to the ongoing discussion on fluoride toxicity.

Since the addition of fluoride to drinking water in the 1940s, there have been frequent and sometimes heated discussions regarding its benefits and risks. In a recently published review, we addressed the question if current exposure levels in Europe represent a risk to human health. This review was discussed in an editorial asking why we did not calculate benchmark doses (BMD) of fluoride neurotoxicity for humans. Here, we address the question, why it is problematic to calculate BMDs based on the currently available data. Briefly, the conclusions of the available studies are not homogeneous, reporting negative as well as positive results; moreover, the positive studies lack control of confounding factors such as the influence of well-known neurotoxicants. We also discuss the limitations of several further epidemiological studies that did not meet the inclusion criteria of our review. Finally, it is important to not only focus on epidemiological studies. Rather, risk analysis should consider all available data, including epidemiological, animal, as well as in vitro studies. Despite remaining uncertainties, the totality of evidence does not support the notion that fluoride should be considered a human developmental neurotoxicant at current exposure levels in European countries.

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.

Toxicity of fluoride: critical evaluation of evidence for human developmental neurotoxicity in epidemiological studies, animal experiments and in vitro analyses.

Recently, epidemiological studies have suggested that fluoride is a human developmental neurotoxicant that reduces measures of intelligence in children, placing it into the same category as toxic metals (lead, methylmercury, arsenic) and polychlorinated biphenyls. If true, this assessment would be highly relevant considering the widespread fluoridation of drinking water and the worldwide use of fluoride in oral hygiene products such as toothpaste. To gain a deeper understanding of these assertions, we reviewed the levels of human exposure, as well as results from animal experiments, particularly focusing on developmental toxicity, and the molecular mechanisms by which fluoride can cause adverse effects. Moreover, in vitro studies investigating fluoride in neuronal cells and precursor/stem cells were analyzed, and 23 epidemiological studies published since 2012 were considered. The results show that the margin of exposure (MoE) between no observed adverse effect levels (NOAELs) in animal studies and the current adequate intake (AI) of fluoride (50 µg/kg b.w./day) in humans ranges between 50 and 210, depending on the specific animal experiment used as reference. Even for unusually high fluoride exposure levels, an MoE of at least ten was obtained. Furthermore, concentrations of fluoride in human plasma are much lower than fluoride concentrations, causing effects in cell cultures. In contrast, 21 of 23 recent epidemiological studies report an association between high fluoride exposure and reduced intelligence. The discrepancy between experimental and epidemiological evidence may be reconciled with deficiencies inherent in most of these epidemiological studies on a putative association between fluoride and intelligence, especially with respect to adequate consideration of potential confounding factors, e.g., socioeconomic status, residence, breast feeding, low birth weight, maternal intelligence, and exposure to other neurotoxic chemicals. In conclusion, based on the totality of currently available scientific evidence, the present review does not support the presumption that fluoride should be assessed as a human developmental neurotoxicant at the current exposure levels in Europe.

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.

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.

Urinary biomarkers of exposure to the mycoestrogen zearalenone and its modified forms in German adults.

Zearalenone (ZEN), a mycotoxin with estrogenic activity, can exert adverse endocrine effects in mammals and is thus of concern for humans. ZEN is found in cereal crops and grain-based foods, often along with modified ('masked') forms usually not detected in routine contaminant analysis, e.g., ZEN-O-ß-glucosides and ZEN-14-sulfate. These contribute to mycoestrogen exposure, as they are cleaved in the gastrointestinal tract to ZEN, and further metabolized in animals and humans to α- and ß-zearalenol (α-ZEL and ß-ZEL). ZEN and its metabolites are mainly excreted as conjugates in urine, allowing to monitor human exposure by a biomarker-based approach. Here, we report on a new study in German adults (n = 60) where ZEN, α-ZEL, and ß-ZEL were determined by LC-MS/MS analysis after enzymatic hydrolysis and immunoaffinity column clean-up of the aglycones in urines. Biomarkers were detected in all samples: ZEN ranges 0.04-0.28 (mean 0.10 ± 0.05; median 0.07) ng/mL; α-ZEL ranges 0.06-0.45 (mean 0.16 ± 0.07; median 0.13) ng/mL, and ß-ZEL ranges 0.01-0.20 (mean 0.05 ± 0.04; median 0.03) ng/mL. Notably, average urinary levels of α-ZEL, the more potent estrogenic metabolite, are higher than those of ZEN, while ß-ZEL (less estrogenic than ZEN) is found at lower levels than the parent mycotoxin. Similar results were found in ten persons who collected multiple urine samples to gain more insight into temporal fluctuations in ZEN biomarker levels; here some urines had higher maximal concentrations of total ZEN (the sum of ZEN, α-ZEL, and ß-ZEL) with 1.6 and 1.01 ng/mL, i.e., more than those found in the majority of other urines. A preliminary approach to translate the new urinary biomarker data into dietary mycotoxin intake suggests that exposure of most individuals in our cohort is probably below the tolerable daily intake (TDI) of 0.25 µg/kg b.w. set by EFSA as group value for ZEN and its modified forms while that of some individuals exceed it. In conclusion, biomonitoring can help to assess consumer exposure to the estrogenic mycotoxin ZEN and its modified forms and to identify persons at higher risk.

Scientific principles for the identification of endocrine-disrupting chemicals: a consensus statement.

Endocrine disruption is a specific form of toxicity, where natural and/or anthropogenic chemicals, known as "endocrine disruptors" (EDs), trigger adverse health effects by disrupting the endogenous hormone system. There is need to harmonize guidance on the regulation of EDs, but this has been hampered by what appeared as a lack of consensus among scientists. This publication provides summary information about a consensus reached by a group of world-leading scientists that can serve as the basis for the development of ED criteria in relevant EU legislation. Twenty-three international scientists from different disciplines discussed principles and open questions on ED identification as outlined in a draft consensus paper at an expert meeting hosted by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany on 11-12 April 2016. Participants reached a consensus regarding scientific principles for the identification of EDs. The paper discusses the consensus reached on background, definition of an ED and related concepts, sources of uncertainty, scientific principles important for ED identification, and research needs. It highlights the difficulty in retrospectively reconstructing ED exposure, insufficient range of validated test systems for EDs, and some issues impacting on the evaluation of the risk from EDs, such as non-monotonic dose-response and thresholds, modes of action, and exposure assessment. This report provides the consensus statement on EDs agreed among all participating scientists. The meeting facilitated a productive debate and reduced a number of differences in views. It is expected that the consensus reached will serve as an important basis for the development of regulatory ED criteria.

[Mycotoxins in food : Occurrence, importance and health risk]. / Mykotoxine in Lebensmitteln : Vorkommen, Bedeutung und gesundheitliches Risiko.

Mycotoxins are produced by a variety of fungal species on crops or stored commodities; they appear as primary or secondary contaminants and via the carryover effect in the food chain. The global occurrence of many mycotoxins with a wide spectrum of toxicities is the subject of interdisciplinary research and a relevant topic for consumer protection. This overview aims to raise interest with sections of the hazardous properties of important mycotoxins (from aflatoxin to zearalenone) and describes recent developments in the area of exposure assessment and basic principles applied in risk characterization. Challenges with regard to new insights about modified and so far unregulated mycotoxins are also addressed.

Biomonitoring of Mycotoxins in Human Breast Milk: Current State and Future Perspectives.

Human breast milk is considered as the best and ideal form of nutrition for infants. However, food contaminants such as mycotoxins, which may be transferred from maternal blood to milk, are poorly described. Mycotoxins are a major group of natural toxins frequently detected in foods. Here, we review the current state-of-the-art in the monitoring of mycotoxins in human breast milk, i.e., knowledge on occurrence, metabolism, and analytical assays utilized for their quantification. We highlight that most of the data captured to date have not been verified with the precision now capable utilizing LC-MS/MS and LC-HRMS approaches. One concern is that some studies may overestimate individual measures, and most cannot capture the patterns and levels of mycotoxin mixtures. We propose accurate assessment as a priority, especially for aflatoxins, fumonisins, ochratoxin A, zearalenone, and deoxynivalenol as well as their major metabolites. However, also so-called emerging toxins such as citrinin, the enniatins, beauvericin, aurofusarin, or Alternaria toxins should be considered to evaluate their potential relevance. Key requirements for analytical quality assurance are identified and discussed to guide future developments in this area. Moreover, research needs including investigations of lactational transfer rates, the role of human metabolism for bioactivation or detoxification, and an evaluation of potential combinatory effects of different mycotoxins are pointed out. It is hoped that LC-MS based multianalyte methods will enable more accurate, rapid and affordable human biomonitoring approaches that support informed decisions for maternal and infant health.

Biomonitoring of the mycotoxin Zearalenone: current state-of-the art and application to human exposure assessment.

Zearalenone (ZEN), a mycotoxin with high estrogenic activity in vitro and in vivo, is a widespread food contaminant that is commonly detected in maize, wheat, barley, sorghum, rye and other grains. Human exposure estimates based on analytical data on ZEN occurrence in various food categories and food consumption data suggest that human exposure to ZEN and modified forms of ZEN may be close to or even exceed the tolerable daily intake (TDI) derived by the European Food Safety Authority (EFSA) for some consumer groups. Considering the inherent uncertainties in estimating dietary intake of ZEN that may lead to an under- or overestimation of ZEN exposure and consequently human risk and current lack of data on vulnerable consumer groups, there is a clear need for more comprehensive and reliable exposure data to refine ZEN risk assessment. Human biomonitoring (HBM) is increasingly being recognized as an efficient and cost-effective way of assessing human exposure to food contaminants, including mycotoxins. Based on animal and (limited) human data on the toxicokinetics of ZEN, it appears that excretion of ZEN and its major metabolites may present suitable biomarkers of ZEN exposure. In view of the limitations of available dietary exposure data on ZEN and its modified forms, the purpose of this review is to provide an overview of recent studies utilizing HBM to monitor and assess human exposure to ZEN. Considerations are given to animal and human toxicokinetic data relevant to HBM, analytical methods, and available HBM data on urinary biomarkers of ZEN exposure in different cohorts.

Urinary biomarkers of ochratoxin A and citrinin exposure in two Bangladeshi cohorts: follow-up study on regional and seasonal influences.

Biomonitoring studies can provide valuable insights into human mycotoxin exposure, especially when food contaminant data are scarce or unavailable as in Bangladesh. First biomonitoring data in Bangladeshi adults indicated exposure to the nephrotoxic mycotoxins ochratoxin A (OTA) and citrinin (CIT). This led us to conduct a follow-up study with analysis of urinary biomarkers for both CIT and OTA to investigate regional and seasonal influences on mycotoxin exposure in two Bangladeshi cohorts. In total, 164 urines were collected (n = 69 in summer, n = 95 in winter) from residents of a rural and an urban area, among which there were 62 participants enrolled in both sampling periods. Most urines had detectable biomarker levels (OTA, CIT and its metabolite dihydrocitrinone, HO-CIT), with more or less pronounced differences with regard to season and region. In both cohorts, OTA was found at a mean level of 0.06 ± 0.10 ng/mL urine (range 0.01-0.55 ng/mL) in summer and a mean of 0.19 ± 0.38 ng/mL (range 0.01-1.75 ng/mL) in winter season. A season difference was significant in the rural cohort, but not in the urban cohort, and slightly higher mean OTA levels in the rural compared to the urban cohort were only observed in winter urines. CIT biomarkers showed more pronounced variations, with a CIT mean of 0.10 ± 0.17 ng/mL (range 0.02-1.22 ng/mL) and HO-CIT mean of 0.42 ± 0.98 ng/mL (range 0.02-5.39 ng/mL) in summer, and CIT mean of 0.59 ± 0.98 ng/mL (range 0.05-5.03 ng/mL) and HO-CIT mean of 3.18 ± 8.49 ng/mL (range 0.02-46.44 ng/mL) in winter urines of both cohorts. In both seasons, total CIT biomarker concentrations were significantly higher in the rural cohort than in the urban cohort. A provisional daily intake for CIT was calculated and exceeded a preliminary value set by EFSA (0.2 µg/kg/d) in 10 and 24 % of participants in summer and winter, respectively. No significant correlations were found between urinary biomarker levels and intake of certain types of food, except for a positive trend for higher rice consumption. Our results in the Bangladeshi population indicate frequent co-exposure to nephrotoxic mycotoxin food contaminants that vary by season and region.

Occurrence of aflatoxin M1 in urines from rural and urban adult cohorts in Bangladesh.

Aflatoxins are important mycotoxins produced by Aspergillus flavus and A. parasiticus, moulds which contaminate mainly grains and nuts, especially in hot and humid climate. Presence of aflatoxin B1 (AFB1), the most toxic one and a potent hepatocarcinogen, has been reported in food and feed in Bangladesh and raised concerns about mycotoxin exposure in the population. Biomonitoring provides the best approach to assess human exposure from various sources and by all routes. Part of the ingested AFB1 is converted in the body to aflatoxin M1 (AFM1), a metabolite that has served as biomarker of AFB1 exposure, as it is excreted in urine, and thus enables non-invasive sampling, a relevant aspect in field studies. This investigation measured the AFM1 concentration in urines collected from adult residents of a rural (n = 52) and an urban (n = 43) area in the Rajshahi district of Bangladesh. The urinary levels of AFM1 were determined by enzyme-linked immunosorbent assay. AFM1 was detected in 46 % of all urine samples at a range of 31-348 pg/mL. The median and mean concentration of AFM1 in urine was 61 and 80 ± 60 pg/mL, respectively. A significant difference (p < 0.05) was found at the mean level of AFM1 between the rural (99 ± 71 pg/mL) and urban (54 ± 15 pg/mL) cohort. Urinary AFM1 levels did not show significant correlations with food frequency data or age, gender and body mass index of the participants. Among them, the highest mean AFM1 level (101 ± 71 pg/mL) was observed in the 50-60 years age group. In conclusion, detection frequency and urinary AFM1 levels in the Bangladeshi adults support concerns regarding their dietary exposure to AFB1. These first data warrant further biomarker-based studies in children and in cohorts of other parts of the country.

Biomonitoring of Mycotoxins in Urine: Pilot Study in Mill Workers.

Contamination of grains with mycotoxins results in a dietary background exposure of the general population. In occupational settings such as during processing of raw materials as in milling, an additional mycotoxin exposure by inhalation is possible. Biomonitoring is an integrative approach to assess human exposure from various sources and by all routes. To investigate possible workplace exposure to mycotoxins, a pilot study was conducted that compared levels of urinary biomarkers in mill workers to those in a control group with dietary mycotoxin intake alone. Workers (n = 17) from three grain mills in North Rhine Westphalia, Germany, provided spot urines during shift; volunteers (n = 13, IfADo staff) with matched age structure served as control group. The mycotoxins selected for biomarker analysis were citrinin (CIT) deoxynivalenol (DON), ochratoxin A (OTA), and zearalenone (ZEN). Immunoaffinity columns (CIT, DON, ZEN) or liquid-liquid extraction (OTA) was employed for urine sample cleanup prior to targeted analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or by high-performance liquid chromatography (HPLC). In addition, mycotoxin metabolites that may be formed in the organism were analyzed, including deepoxy-deoxynivalenol (DOM-1), ochratoxin alpha (OTα), dihydrocitrinone (DH-CIT), and α- and ß-zearalenol (α- and ß-ZEL), as well as phase II metabolites that were hydrolyzed with ß-glucuronidase/arylsulfatase prior to sample cleanup. All analyte concentrations were adjusted for creatinine (crea) content in the spot urine samples. Citrinin, DON, OTA, and ZEN were detected in nearly all urine samples from mill workers and controls. Interestingly, DH-CIT was found at higher mean levels than the parent compound (~0.14 and 0.045 µg/g crea, respectively), suggesting an effective metabolism of CIT in humans. Other metabolites DOM-1, OTα, and α- and ß-ZEL were detected less frequently in urine. Deoxynivalenol was detected at the highest concentrations (mean ~6 µg/g crea), followed by OTA (mean ~0.08 µg/g crea); ZEN (mean ~0.03 µg/g crea) and its metabolites appeared in urine at lower levels. Mycotoxin biomarker levels in urine from mill workers and controls were not significantly different. From these results it is concluded that biomarker levels measured in urine samples from the two cohorts reflect mainly dietary mycotoxin exposure. An additional occupational (inhalational) exposure of mill workers, if any, is apparently low at the investigated workplaces.

Opinion of the Scientific Committee on Consumer safety (SCCS) - Opinion on the safety of the use of deoxyarbutin in cosmetic products.

CONCLUSION OF THE OPINION: Although on the basis of the provided scientific data the use of deoxyarbutin as such can be considered safe for consumers in cosmetic products in a concentration up to 3% in face creams, hydroquinone will be formed at levels which raise concerns with regard to the safety of such products during life-cycle of the product (e.g. storage conditions and stability under in-use conditions). Therefore, the overall conclusion of the SCCS is that the use of deoxyarbutin up to 3% in face creams is not safe.