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.

Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters.

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.

Commentary of the SKLM to the EFSA opinion on risk assessment of N-nitrosamines in food.

Dietary exposure to N-nitrosamines has recently been assessed by the European Food Safety Authority (EFSA) to result in margins of exposure that are conceived to indicate concern with respect to human health risk. However, evidence from more than half a century of international research shows that N-nitroso compounds (NOC) can also be formed endogenously. In this commentary of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG), the complex metabolic and physiological biokinetics network of nitrate, nitrite and reactive nitrogen species is discussed with emphasis on its influence on endogenous NOC formation. Pioneering approaches to monitor endogenous NOC have been based on steady-state levels of N-nitrosodimethylamine (NDMA) in human blood and on DNA adduct levels in blood cells. Further NOC have not been considered yet to a comparable extent, although their generation from endogenous or exogenous precursors is to be expected. The evidence available to date indicates that endogenous NDMA exposure could exceed dietary exposure by about 2-3 orders of magnitude. These findings require consolidation by refined toxicokinetics and DNA adduct monitoring data to achieve a credible and comprehensive human health risk assessment.

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.

Antagonistic properties against Fusarium sporotrichioides and glycosylation of HT-2 and T-2 toxins by selected Trichoderma strains.

The present study assessed the ability of Trichoderma to combat F. sporotrichioides, focusing on their antagonistic properties. Tests showed that Trichoderma effectively inhibited F. sporotrichioides mycelial growth, particularly with T. atroviride strains. In co-cultures on rice grains, Trichoderma almost completely reduced the biosynthesis of T-2 and HT-2 toxins by Fusarium. T-2 toxin-α-glucoside (T-2-3α-G), HT-2 toxin-α-glucoside (HT-2-3α-G), and HT-2 toxin-ß-glucoside (HT-2-3ß-G) were observed in the common culture medium, while these substances were not present in the control medium. The study also revealed unique metabolites and varying metabolomic profiles in joint cultures of Trichoderma and Fusarium, suggesting complex interactions. This research offers insights into the processes of biocontrol by Trichoderma, highlighting its potential as a sustainable solution for managing cereal plant pathogens and ensuring food safety.

Incorporation of the histone variant H2A.Z counteracts gene silencing mediated by H3K27 trimethylation in Fusarium fujikuroi.

BACKGROUND: Fusarium fujikuroi is a pathogen of rice causing diverse disease symptoms such as 'bakanae' or stunting, most likely due to the production of various natural products (NPs) during infection. Fusaria have the genetic potential to synthesize a plethora of these compounds with often diverse bioactivity. The capability to synthesize NPs exceeds the number of those being produced by far, implying a gene regulatory network decisive to induce production. One such regulatory layer is the chromatin structure and chromatin-based modifications associated with it. One prominent example is the exchange of histones against histone variants such as the H2A variant H2A.Z. Though H2A.Z already is well studied in several model organisms, its regulatory functions are not well understood. Here, we used F. fujikuroi as a model to explore the role of the prominent histone variant FfH2A.Z in gene expression within euchromatin and facultative heterochromatin. RESULTS: Through the combination of diverse '-omics' methods, we show the global distribution of FfH2A.Z and analyze putative crosstalks between the histone variant and two prominent histone marks, i.e., H3K4me3 and H3K27me3, important for active gene transcription and silencing, respectively. We demonstrate that, if FfH2A.Z is positioned at the + 1-nucleosome, it poises chromatin for gene transcription, also within facultative heterochromatin. Lastly, functional characterization of FfH2A.Z overexpression and depletion mutants revealed that FfH2A.Z is important for wild type-like fungal development and secondary metabolism. CONCLUSION: In this study, we show that the histone variant FfH2A.Z is a mark of positive gene transcription and acts independently of the chromatin state most likely through the stabilization of the + 1-nucleosome. Furthermore, we demonstrate that FfH2A.Z depletion does not influence the establishment of both H3K27me3 and H3K4me3, thus indicating no crosstalk between FfH2A.Z and both histone marks. These results highlight the manifold functions of the histone variant FfH2A.Z in the phytopathogen F. fujikuroi, which are distinct regarding gene transcription and crosstalk with the two prominent histone marks H3K27me3 and H3K4me3, as proposed for other model organisms.

Structural Impact of Steroidal Glycoalkaloids: Barrier Integrity, Permeability, Metabolism, and Uptake in Intestinal Cells.

SCOPE: Potato tubers represent an essential food component all over the world and an important supplier of carbohydrates, fiber, and valuable proteins. However, besides their health promoting effects, potatoes contain α-solanine and α-chaconine, which are toxic steroidal glycoalkaloids (SGAs). Other solanaceous plants like eggplants and tomatoes produce SGAs as well, different in their chemical structure. This study aims to investigate toxic effects (cholinesterase inhibition, membrane, and barrier disruption), permeability, metabolism, and structure-activity relationships of SGAs. METHODS AND RESULTS: α-solanine, α-chaconine, α-solasonine, α-solamargine, α-tomatine, and their respective aglycones solanidine, solasodine, and tomatidine are analyzed using Ellman assay, cellular impedance spectroscopy, cell extraction, and Caco-2 intestinal model. Additionally, metabolism is analyzed by HPLC-MS techniques. The study observes dependencies of barrier disrupting potential and cellular uptake on the carbohydrate moiety of SGAs, while permeability and acetylcholinesterase (AChE) inhibition are dominated by the steroid backbone. SGAs show low permeabilities across Caco-2 monolayers in subtoxic concentrations. In contrast, their respective aglycones reveal higher permeabilities, but are extensively metabolized. CONCLUSION: Besides structure-activity relationships, this study provides new information on the overall effects of steroidal alkaloids on intestinal cells and closes a gap of knowledge for the metabolic pathway from oral uptake to final excretion.

Co-occurrence of T-2 and HT-2 Mycotoxins and α and ß Anomers of Their Glucosides in Wheat and Oat Grains.

The aim of this study was to simultaneously determine T-2 and HT-2 toxins and the α and ß anomers of their glucosides to assess their content in wheat and oat grains harvested in Poland (2020-2022). Of 298 wheat samples, only 14 (5%) contained the sum of the T-2 and HT-2 toxins (average 34.2 µg/kg; 10.6-67.7 µg/kg). In oat (n = 129), these compounds were detected much more frequently (70% of samples) at an average level of 107.5 µg/kg (6.9-949.1 µg/kg). The sum of T-2 and HT-2 glucosides was detectable in 3% of the wheat (average 16.3 µg/kg; 7.1-39.4 µg/kg) and 65% of the oat samples (average 35.1 µg/kg; 4.0-624.1 µg/kg). Following the study, T-2-3-α-glucoside was identified as the only naturally occurring anomer, while both anomers of HT-2-3-glucosides were detected with higher contents and occurrence rates of HT-2-3-ß-glucoside than the α anomer of this compound.

H3K27me3 is vital for fungal development and secondary metabolite gene silencing, and substitutes for the loss of H3K9me3 in the plant pathogen Fusarium proliferatum.

Facultative heterochromatin marked by histone H3 lysine 27 trimethylation (H3K27me3) is an important regulatory layer involved in secondary metabolite (SM) gene silencing and crucial for fungal development in the genus Fusarium. While this histone mark is essential in some (e.g., the rice pathogen Fusarium fujikuroi), it appears dispensable in other fusaria. Here, we show that deletion of FpKMT6 is detrimental but not lethal in the plant pathogen Fusarium proliferatum, a member of the Fusarium fujikuroi species complex (FFSC). Loss of FpKmt6 results in aberrant growth, and expression of a large set of previously H3K27me3-silenced genes is accompanied by increased H3K27 acetylation (H3K27ac) and an altered H3K36me3 pattern. Next, H3K9me3 patterns are affected in Δfpkmt6, indicating crosstalk between both heterochromatic marks that became even more obvious in a strain deleted for FpKMT1 encoding the H3K9-specific histone methyltransferase. In Δfpkmt1, all H3K9me3 marks present in the wild-type strain are replaced by H3K27me3, a finding that may explain the subtle phenotype of the Δfpkmt1 strain which stands in marked contrast to other filamentous fungi. A large proportion of SM-encoding genes is allocated with H3K27me3 in the wild-type strain and loss of H3K27me3 results in elevated expression of 49% of them. Interestingly, genes involved in the biosynthesis of the phytohormones gibberellins (GA) are among the most upregulated genes in Δfpkmt6. Although several FFSC members harbor GA biosynthetic genes, its production is largely restricted to F. fujikuroi, possibly outlining the distinct lifestyles of these notorious plant pathogens. We show that H3K27me3 is involved in GA gene silencing in F. proliferatum and at least one additional FFSC member, and thus, may serve as a regulatory layer for gene silencing under non-favoring conditions.

Evaluation of Food Intake Biomarkers for Red Bell Peppers in Human Urine Based on HPLC-MS/MS Analysis.

SCOPE: The validation of dietary biomarkers is essential for the use in objective and quantitative assessment of the human dietary intake. In this study, the urinary excretion of previously identified potential biomarkers after intake of red bell peppers is analyzed. METHODS AND RESULTS: The urine samples obtained after a two-phase dietary intervention study in which 14 volunteers participated are quantitatively analyzed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) after an extensive validation. In the first phase, the volunteers abstain completely from bell peppers and paprika products (control group) and in the second phase, the volunteers consume a defined amount of fresh red bell peppers (case group). After analysis, all potential biomarkers show high dispersions of their concentration, indicating interindividual differences. The glucuronidated apocarotenoid (compound 1), which probably resulted from the main carotenoids of red Capsicum fruits, shows a rapid urinary excretion. The other glucuronidated metabolites (compounds 2-8), described as potential derivatives of capsianosides from Capsicum, show a slightly delayed but longer urinary excretion. CONCLUSIONS: A correlation between an intake of red bell pepper and the urinary excretion of recently described potential biomarkers is observed. Due to large interindividual differences, it is reasonable to assume that at least the qualitative detection of the consumption of bell peppers and possibly all Capsicum fruits is feasible.

Acetaldehyde as a Food Flavoring Substance: Aspects of Risk Assessment.

The Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) has reviewed the currently available data in order to assess the health risks associated with the use of acetaldehyde as a flavoring substance in foods. Acetaldehyde is genotoxic in vitro. Following oral intake of ethanol or inhalation exposure to acetaldehyde, systemic genotoxic effects of acetaldehyde in vivo cannot be ruled out (induction of DNA adducts and micronuclei). At present, the key question of whether acetaldehyde is genotoxic and mutagenic in vivo after oral exposure cannot be answered conclusively. There is also insufficient data on human exposure. Consequently, it is currently not possible to reliably assess the health risk associated with the use of acetaldehyde as a flavoring substance. However, considering the genotoxic potential of acetaldehyde as well as numerous data gaps that need to be filled to allow a comprehensive risk assessment, the SKLM considers that the use of acetaldehyde as a flavoring may pose a safety concern. For reasons of precautionary consumer protection, the SKLM recommends that the scientific base for approval of the intentional addition of acetaldehyde to foods as a flavoring substance should be reassessed.

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.

Optimization of sample clean-up for the determination of small amounts of MOSH and MOAH in edible oils - method DGF C-VI 22 (20).

Methods for determining MOSH and MOAH in edible oils showed major problems with interlaboratory comparability of analytical results, especially in the lower concentration range below 10 mg/kg. However, a method with improved sensitivity and reproducibility is urgently needed to obtain a valid data basis for minimization efforts. To cope this problem a new method was created in 2020. The method was established as the standard method DGF C-VI 22 (20) of the German Society for Fat Science e.V. (DGF). For the development of this method different sample epoxidation approaches have been performed, evaluated and improved. Additionally, a saponification, a decision tree for sample preparation, an upstream clean-up column and a system suitability test were introduced. The focus was on reliability and interlaboratory comparability over all edible oil matrices up to a LOQ of 1 mg/kg. The optimized method was validated in terms of trueness and precision in a collaborative trail with 11 laboratories. The achieved recovery rates of 89-105% MOSH and 70-105% MOAH met the JRC requirements. Method and validation results were obtained with HorRat values between 1.3 and 1.8 for MOSH and MOAH.

Polyphenols Cause Structure Dependent Effects on the Metabolic Profile of Human Hepatocarcinogenic Cells.

SCOPE: Although many beneficial health effects are attributed to polyphenols their influence on the human metabolome has not been elucidated yet. The ubiquitous occurrence of polyphenols in the human diet demands comprehensive knowledge about physiological and toxicological effects of these compounds on human cells. METHODS AND RESULTS: The human hepatocarcinogenic cell line HepG2 is used to elucidate the effects of 13 polyphenols and three respective phenolic degradation products on the human metabolome using HPLC-MS/MS. To investigate structure-activity-relationships, structurally related examples of polyphenols from different compound classes are selected. The analysis of catechins points toward a relation between the degree of hydroxylation and the extent of metabolic effects particularly on the urea cycle and the pentose phosphate pathway (PPP). A correlation between the modulation of the PPP and the stability of the compounds is demonstrated, which may be caused by reactive oxygen species (ROS). The incubation of flavones and alkenylbenzenes demonstrates reduced activity of methoxylated compounds and no impact of the B-ring position. CONCLUSION: In general, polyphenols induce a multitude of metabolic effects, for example, on energy metabolism, PPP, and urea cycle. These metabolic alterations may be related to the widely reported bioactivity of these compounds such as the anticarcinogenic effects.

Dose-dependent expression of GFI1 alters metabolism in the haematopoietic progenitors and MLL::AF9-induced leukaemic cells.

Growth factor independence 1 (GFI1) is a transcriptional repressor protein that plays an essential role in the differentiation of myeloid and lymphoid progenitors. We and other groups have shown that GFI1 has a dose-dependent role in the initiation, progression, and prognosis of acute myeloid leukaemia (AML) patients by inducing epigenetic changes. We now demonstrate a novel role for dose-dependent GFI1 expression in regulating metabolism in haematopoietic progenitor and leukaemic cells. Using in-vitro and ex-vivo murine models of MLL::AF9-induced human AML and extra-cellular flux assays, we now demonstrate that a lower GFI1 expression enhances oxidative phosphorylation rate via upregulation of the FOXO1- MYC axis. Our findings underscore the significance of therapeutic exploitation in GFI1-low-expressing leukaemia cells by targeting oxidative phosphorylation and glutamine metabolism.

Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA.

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell's metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme's function from transcription to replication.

Degradation products of aflatoxin M1 (AFM1) formed by high voltage atmospheric cold plasma (HVACP) treatment.

Cold plasma technology is a novel non-thermal technology that has shown promising results for food decontamination and improving food safety. This study is a continuation of a previous investigation of the treatment of AFM1-contaminated skim and whole milk samples by HVACP. Previous research has shown HVACP is effective in degrading aflatoxin M1 (AFM1) in milk. The goal of this study is to identify the degradation products of AFM1 after HVACP treatment in pure water. An HVACP direct treatment at 90 kV using modified air (MA65: 65% O2, 30% CO2, 5% N2) was performed for up to 5 min at room temperature on a 5.0 mL water sample in a Petri dish artificially contaminated with 2 µg/mL of AFM1. The degradants of AFM1 were analyzed and their molecular formulae were elucidated by using high-performance liquid-chromatography time-of-flight mass spectrometry (HPLC-TOF-MS). Three main degradation products were observed and based on mass spectrometric fragmentation pathways, chemical structures for the degradation products were tentatively assigned. According to the structure-bioactivity relationship of AFM1, the bioactivity of the AFM1 samples treated with HVACP was reduced due to the disappearance of the C8-C9 double bond in the furofuran ring in all of the degradation products.

Intestinal Metabolism of Selected Steroidal Glycoalkaloids in the Pig Cecum Model.

Due to the presence of the steroidal glycoalkaloid solanine, the potato was chosen as Germany's poisonous plant of the year 2022. Steroidal glycoalkaloids are secondary plant metabolites which have been reported to induce toxic as well as beneficial health effects. Nevertheless, data regarding occurrence, toxicokinetics, and metabolism of steroidal glycoalkaloids is scarce, and substantially more research is required for a proper risk assessment. Therefore, the intestinal metabolism of solanine, chaconine, solasonine, solamargine, and tomatine was investigated using the ex vivo pig cecum model. All steroidal glycoalkaloids were degraded by the porcine intestinal microbiota, releasing the respective aglycon. Furthermore, the hydrolysis rate was strongly dependent on the linked carbohydrate side chain. Solanine and solasonine, which are linked to a solatriose, were metabolized significantly faster than the chaconine and solamargin, which are linked to a chacotriose. In addition, stepwise cleavage of the carbohydrate side chain and the formation of ß- and γ-intermediates were detected by HPLC-HRMS. The results provide valuable insights into the intestinal metabolism of selected steroidal glycoalkaloids and help to reduce uncertainties and improve risk assessment.

Maternal exposure to multiple mycotoxins and adverse pregnancy outcomes: a prospective cohort study in rural Bangladesh.

There is limited and inconsistent evidence, primarily from cross-sectional studies, linking mycotoxins to adverse birth outcomes. This study investigates the potential role of maternal dietary exposure to multiple mycotoxins in the development of several adverse pregnancy and birth outcomes. We analyzed data from 436 singleton pregnancies enrolled in a prospective cohort study in the rural Habiganj district, Bangladesh, between July 2018 and November 2019. Thirty-five urinary mycotoxin biomarkers were quantified using liquid chromatography coupled with tandem mass spectrometry and used to estimate dietary mycotoxin exposure. Multivariable regression models, adjusted for potential confounding and clustering, were fitted to assess the associations between maternal exposure to frequently occurring mycotoxins (ochratoxin A-OTA, citrinin- CIT, and Deoxynivalenol- DON) and pregnancy loss, preterm birth (PTB), low birth weight (LBW), born small-for-gestational-age (SGA) and small-vulnerable newborn. The results indicate that only in 16 of 436 pregnancies (4%) were urine samples free from all investigated mycotoxins. Biomarkers for six major mycotoxins were detected in the urine samples. OTA (95%), CIT (61%), and DON (6%) were most frequently detected, with at least two mycotoxins co-occurring in the majority of women (63%). There was evidence that maternal dietary intake of OTA was associated with higher odds of having an LBW baby, with the odds increasing in a dose-dependent manner. We found no evidence of associations between pregnancy loss, PTB, SGA, small-vulnerable newborns, and maternal dietary exposure to OTA, CIT, and DON, albeit with large confidence intervals, so findings are consistent with protective as well as large harmful effects. Exposure to multiple mycotoxins during pregnancy is widespread in this rural community and represents a health risk for mothers and babies. Tailored public health policies and interventions must be implemented to reduce mycotoxin exposure to the lowest possible level.