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.

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.

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.

Dietary glycation compounds - implications for human health.

The term "glycation compounds" comprises a wide range of structurally diverse compounds that are formed endogenously and in food via the Maillard reaction, a chemical reaction between reducing sugars and amino acids. Glycation compounds produced endogenously are considered to contribute to a range of diseases. This has led to the hypothesis that glycation compounds present in food may also cause adverse effects and thus pose a nutritional risk to human health. In this work, the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) summarized data on formation, occurrence, exposure and toxicity of glycation compounds (Part A) and systematically assessed potential associations between dietary intake of defined glycation compounds and disease, including allergy, diabetes, cardiovascular and renal disease, gut/gastrotoxicity, brain/cognitive impairment and cancer (Part B). A systematic search in Pubmed (Medline), Scopus and Web of Science using a combination of keywords defining individual glycation compounds and relevant disease patterns linked to the subject area of food, nutrition and diet retrieved 253 original publications relevant to the research question. Of these, only 192 were found to comply with previously defined quality criteria and were thus considered suitable to assess potential health risks of dietary glycation compounds. For each adverse health effect considered in this assessment, however, only limited numbers of human, animal and in vitro studies were identified. While studies in humans were often limited due to small cohort size, short study duration, and confounders, experimental studies in animals that allow for controlled exposure to individual glycation compounds provided some evidence for impaired glucose tolerance, insulin resistance, cardiovascular effects and renal injury in response to oral exposure to dicarbonyl compounds, albeit at dose levels by far exceeding estimated human exposures. The overall database was generally inconsistent or inconclusive. Based on this systematic review, the SKLM concludes that there is at present no convincing evidence for a causal association between dietary intake of glycation compounds and adverse health effects.

Considering the implication of endogenous glycation compounds in aging and disease, dietary exposure via consumption of an "AGE (advanced glycation end product) rich diet" is increasingly suggested to pose a potential health risk. However, studies attempting to assess an association between dietary glycation compounds and adverse health effects frequently suffer from insufficient chemical analysis of glycation compounds, including inadequate structural characterization and limited quantitative data. The Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) previously defined quality criteria for studies designed to assess the effects of dietary glycation compounds on human health. The aim of the present work is to summarize data on formation, occurrence, exposure and toxicity of glycation compounds (Part A) and to systematically evaluate if the currently available scientific database allows for a conclusive assessment of potential health effects of defined glycation compounds (Part B).The term "glycation compounds" comprises a wide range of structurally diverse compounds that derive from the Maillard reaction, a chemical reaction between reducing carbohydrates and amino compounds that occurs during food processing. In the first stage of the Maillard reaction, reducing sugars such as glucose and fructose react for instance with the ε-amino group of lysine, which is most abundant in food ("glycation" of lysine). Subsequently, these primary reaction products undergo Amadori rearrangement to yield products (ARP) such as fructosyllysine (FL) from glucose and also Heyns rearrangement products (HRPs) such as glucosyl- and mannosyllysine from fructose. While ARPs are rapidly formed during food processing, they are not stable and undergo degradation reactions, predominantly to 1,2-dicarbonyl compounds such as glyoxal (GO), methylglyoxal (MGO) and 3-deoxyglucosone (3-DG), which are highly reactive. The last stage of the Maillard reaction is characterized predominantly by the reaction of these dicarbonyl compounds with nucleophilic groups of proteins. The side-chains of lysine and arginine residues as well as the N-termini of proteins are important reaction sites. Carboxyalkylated amino acids such as N-ε-carboxymethyllysine (CML) and N-ε-carboxyethyllysine (CEL) result from reaction of the ε-amino group of lysine with the dicarbonyl compounds GO and MGO. Dicarbonyl compounds with C5 or C6 chains can form cyclic pyrrole derivatives at the ε-amino group of lysine. The most important example for this reaction is pyrraline, which is formed from reaction of 3-DG and lysine. The reaction of dicarbonyl compounds with the guanidino group of arginine mainly leads to hydroimidazolones, of which the MGO-derived hydroimidazolone 1 (MG-H1) is best described in food systems.ARPs are the most abundant glycation products found in food. Up to 55% of the lysine residues in food may be modified to ARPs at the side-chain. Food items particularly rich in ARPs include bread, rusk, biscuits, chocolate, and powdered infant formulas. Exposure estimates range between 0.6­1.6 mg/kg body weight (bw), although exposure may be as high as 14.3 mg/kg bw in individuals consuming foods with extreme ARP concentrations. Foods particularly rich in dicarbonyl compounds include heat-treated or long-term stored items rich in reducing sugars such as jams, alternative sweeteners, soft drinks, honey, candies, cookies, and vinegars, especially balsamico-type vinegars. The main contributors to the daily intake of MGO, GO, and 3-DG are coffee and bread. Dietary exposure to dicarbonyl compounds has been estimated to range between 0.02­0.29 mg/kg bw/d for MGO, 0.04­0.16 mg/kg bw/d for GO, 0.14­2.3 mg/kg bw/d for 3-DG, and 0.08­0.13 mg/kg bw/d for 3-deoxygalactosone (3-DGal). Dietary intake of 5-hydroxymethylfurfural (HMF), which can be formed from 3-DG, is estimated to range between 0.0001­0.9 mg/kg bw/d. Exposure estimates for individual glycated amino acids range from 0.03­0.35 mg/kg bw/d for CML, 0.02­0.04 mg/kg bw/d for CEL and 0.19­0.41 mg/kg bw/d for MG-H1. From a model diet consisting of 1 L milk, 500 g bakery products and 400 mL coffee, an intake of pyrraline corresponding to 0.36 mg/kg bw/d for a 70 kg person was estimated.Quantitative analysis of individual glycation compounds or their metabolites in tissues or body fluids as well as their reaction products with amino acids, proteins or DNA may serve to monitor exposure to glycation compounds. However, since glycation compounds are also formed endogenously, these biomarkers reflect the totality of the exposure, making it inherently difficult to define the body burden due to dietary intake against the background of endogenous formation.Information on the toxicokinetics and toxicity of glycation compounds is scarce and mostly limited to the reactive dicarbonyl compounds GO, MGO, 3-DG, HMF, and individual glycated amino acids such as CML and CEL. Acute toxicity of dicarbonyl compounds is low to moderate. There are some data to suggest that rapid detoxification of dicarbonyls in the gastrointestinal tract and liver may limit their oral bioavailability. Biotransformation of GO and MGO occurs predominantly via the glutathione (GSH)-dependent glyoxalase system, and to a lesser extent via glutathione-independent aldo-keto-reductases, which are also responsible for biotransformation of 3-DG. GO, MGO and 3-DG readily react with DNA bases in vitro, giving rise to DNA adducts. There is clear evidence for genotoxicity of GO, MGO and 3-DG. Repeated dose toxicity studies on GO consistently reported reduced body weight gain concomitant with reduced food and water consumption but did not identify compound related changes in clinical chemistry and hematology or histopathological lesions. There is also no evidence for systemic carcinogenicity of GO and MGO based on the available studies. However, initiation/promotion studies indicate that oral exposure to GO may exhibit genotoxic and tumor promoting activity locally in the gastrointestinal tract. From a 2-year chronic toxicity and carcinogenicity study in rats, a NOAEL for systemic toxicity of GO administered via drinking water of 25 mg/kg bw was reported based on reduced body weight and erosions/ulcer in the glandular stomach. Other non-neoplastic and neoplastic lesions were not observed. Acute toxicity of HMF is also low. From a 90-day repeated dose toxicity study in mice, a NOAEL of 94 mg/kg bw was derived based on cytoplasmic alterations of proximal tubule epithelial cells of the kidney. HMF was mostly negative in in vitro genotoxicity tests, although positive findings for mutagenicity were obtained under conditions that promote formation of the chemically reactive sulfuric acid ester 5-sulfoxymethylfurfural. There is some evidence of carcinogenic activity of HMF in female B6C3F1 mice based on increased incidences of hepatocellular adenoma, but not in male mice and rats of both sexes. Although data on oral bioavailability of glycated amino acids are mostly limited to CML, it appears that glycated amino acids may be absorbed from the gastrointestinal tract after oral exposure to their free and protein bound form. Glycated amino acids that are not absorbed in the intestine may be subject to metabolism by the gut microbiome. Glycated amino acids present in the systemic circulation are rapidly eliminated via the urine. Acute oral toxicity of CML is low. Studies in mice and rats reported changes in clinical chemistry parameters indicative of impaired renal and hepatic function. However, these changes were not dose-related and not supported by histopathological evaluation.Previous risk assessments of individual glycation compounds did not identify a health concern at estimated human exposures (GO, HMF) but also noted the lack of data to draw firm conclusions on health risks associated with exposure to MGO.To identify potential associations between dietary intake of defined glycation compounds and disease a systematic review was carried out according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) model, applying the quality criteria previously defined by the SKLM. Using a combination of keywords defining individual glycation compounds and relevant disease patterns linked to the subject area of food, nutrition and diet, a systematic search in Pubmed (Medline), Scopus and Web of Science was performed. Although the present systematic review identified numerous studies that investigated an association between an "AGE-rich diet" and adverse health effects, only a subset of studies was found to comply with the quality criteria defined by the SKLM and was thus considered suitable to assess potential health risks of dietary glycation compounds.For each adverse health effect considered in this assessment, only limited numbers of human studies were identified. Although studies in humans offer the advantage of investigating effects at relevant human exposures, these studies did not provide compelling evidence for adverse effects of dietary glycation compounds. Animal studies identified in this systematic review provide some evidence for induction of impaired glucose tolerance, insulin resistance, cardiovascular effects and renal injury in response to oral exposure to GO and MGO as representatives of dicarbonyl compounds. Only limited evidence points to a link between high intake of glycated amino acids and metabolic disorders. However, these effects were typically reported to occur at dose levels that exceed human dietary exposure, often by several orders of magnitude. Unfortunately, most studies employed only one dose level, precluding characterization of dose-response and derivation of a point of departure for riskassessment. While in vitro studies provide some evidence for a potential mechanistic link between individual glycation compounds and presumed adverse health effects, the clinical and toxicological relevance of the in vitro findings is often limited by the use of high concentrations of glycation compounds that by far exceed human dietary exposure and by insufficient evidence for corresponding adverse effects in vivo. A key question that has not been adequately considered in most studies investigating systemic effects of glycation compounds is the extent of oral bioavailability of dietary glycation compounds, including the form in which MRPs may be taken up (e.g. free vs. peptide bound glycated amino acids). Understanding how much dietary glycation compounds really add to the significant endogenous background is critical to appraise the relevance of dietary MRPs for human health.While it appears mechanistically plausible that glycation of dietary allergens may affect their allergenic potential, the currently available data do not support the hypothesis that dietary glycation compounds may increase the risk for diet-induced allergies. There are no human studies addressing the immunological effects of dietary AGEs. Accordingly, there are no data on whether dietary AGEs promote the development of allergies, nor whether existing allergies are enhanced or attenuated. In numerous in vitro studies, the IgG/E binding ability of antigens and therefore their allergenic potential has been predominantly reported to be reduced by glycation. However, some in vitro studies showed that glycated proteins bind to receptors of immunological cells, and thus may have promoting effects on immune response and inflammation.Although experimental data from animal studies provide some evidence that high doses of individual glycation compounds such as MGO and protein-bound CML may produce certain adverse health effects, including diabetogenic, cardiovascular, metabolic and renal effects, the doses required to achieve these effects by far exceed human dietary exposures. Of note, in the only long-term study identified, a high dose of MGO administered via drinking water to mice for 18 months had no adverse effects on the kidneys, cardiovascular system, or development of diabetes.Experimental data from animal studies provide evidence that high doses of defined glycation compounds such as MGO or protein-bound CML may affect glucose homeostasis. However, the doses required to produce these effects markedly exceed human dietary exposure. Results from human studies are inconclusive: Three short-term intervention studies suggested that diets rich in AGEs may impair glucose homeostasis, whereas one recent intervention study and two observational studies failed to show such an effect.For the cardiovascular system, there is some evidence from in vitro and in vivo studies that high concentrations of MRPs, well above the dietary exposure of humans, may enhance inflammation in the cardiovascular system, induce endothelial damage, increase blood pressure and increase the risk of thrombosis. Only a limited number of human intervention studies investigated potential effects of short-term exposure and longer-term effects of glycation compounds on the cardiovascular system, and yielded inconsistent results. The few observational studies available either found no association between dietary MRP intake and cardiovascular function or even reported beneficial effects. Therefore, currently no definitive conclusion on potential acute and chronic effects of dietary MRPs on inflammation and cardiovascular function can be drawn. However, there is currently also no convincing evidence that potential adverse effects on the cardiovascular system are triggered by dietary MRP intake.Furthermore, human studies did not provide evidence for an adverse effect of dietary MRPs on kidney function. In animal studies with high levels of oral intake, MGO was reported to cause structural and functional effects in the kidney. Several studies show that the concentration of modified proteins and amino acids, such as CML, increases significantly in kidney tissue after oral intake. One study showed a negative effect of a high-temperature-treated diet containing increased CML concentrations on kidney structure integrity and impaired glomerular filtration. The causative relationship of accumulation of dietary MRPs and a functional decline of the kidneys, however, needs further confirmation.With regard to gut health, there is some evidence for alterations in gut microflora composition and the production of individual short-chain fatty acids (SCFAs) upon dietary exposure to glycation compounds. However, this has not been linked to adverse health effects in humans and may rather reflect adaptation of the gut microbiota to changing nutrients. In particular, a human observational study and several animal studies did not find a correlation between the intake of glycation compounds and increased intestinal inflammation. In animal studies, positive effects of glycation compounds on gut tissue damage and dysbiosis during colitis were described.Considering clear evidence for DNA reactivity and genotoxicity of the dicarbonyl compounds GO, MGO and 3-DG, it is plausible to suspect that dicarbonyl compounds may induce mutations and cancer. Although there is some evidence for tumor promoting activity of GO locally in the gastrointestinal tract, the only guideline-compatible chronic rodent bioassays reported erosions and ulcer in the glandular stomach but no treatment-related neoplastic lesions. A recent multinational cohort study with focus on CEL, CML, and MG-H1 found no evidence to support the hypothesis that dietary AGEs are linked to cancer risk.Evidence for an association between human exposure to dietary glycation compounds and detrimental effects on the brain and on cognitive performance is far from being compelling. No human studies fully complying with the defined quality criteria were identified. A few experimental studies reported neuroinflammation and cognitive impairment following dietary MRP exposure, but these can be considered indicative at best and do not support firm conclusions for human health. In addition to utilizing exceedingly high dosages of individual agents like CML, harsh processing conditions causing a multitude of major process-related changes do not allow to convincingly reconcile effects observed with measured/supposed contents of free and protein-bound CML alone.Overall, although dietary glycation compounds have been claimed to contribute to a wide range of adverse health effects, the present critical evaluation of the literature allows the conclusion that the available data are insufficient, inadequate or inconclusive and do not compellingly support the hypothesis of human health risks being related to the presence of glycation compounds in food. The study limitations detailed above, together with the fact that a large number of studies did not comply with the defined quality criteria and therefore had to be excluded highlight the importance of performing adequately designed human or animal studies to inform scientifically reliable health risk assessment.To achieve this, high quality, dependable scientific cooperation within various disciplines is pivotal.

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.

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.

Controversy on health-based guidance values for bisphenol A-the need of criteria for studies that serve as a basis for risk assessment.

Since 2006, the responsible regulatory bodies have proposed five health-based guidance values (HBGV) for bisphenol A (BPA) that differ by a factor of 250,000. This range of HBGVs covers a considerable part of the range from highly toxic to relatively non-toxic substances. As such heterogeneity of regulatory opinions is a challenge not only for scientific risk assessment but also for all stakeholders, the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) analyzed the reasons for the current discrepancy and used this example to suggest improvements for the process of HBGV recommendations. A key aspect for deriving a HBGV is the selection of appropriate studies that allow the identification of a point of departure (PoD) for risk assessment. In the case of BPA, the HBGV derived in the 2023 EFSA assessment was based on a study that reported an increase of Th17 cells in mice with a benchmark dose lower bound (BMDL40) of 0.53 µg/kg bw/day. However, this study does not comply with several criteria that are important for scientific risk assessment: (1) the selected end-point, Th17 cell frequency in the spleen of mice, is insufficiently understood with respect to health outcomes. (2) It is unclear, by which mechanism BPA may cause an increase in Th17 cell frequency. (3) It is unknown, if an increase of Th17 cell frequency in rodents is comparably observed in humans. (4) Toxicokinetics were not addressed. (5) Neither the raw data nor the experimental protocols are available. A further particularly important criterion (6) is independent data confirmation which is not available in the present case. Previous studies using other readouts did not observe immune-related adverse effects such as inflammation, even at doses orders of magnitude higher than in the Th17 cell-based study. The SKLM not only provides here key criteria for the use of such studies, but also suggests that the use of such a "checklist" requires a careful and comprehensive scientific judgement of each item. It is concluded that the Th17 cell-based study data do not represent an adequate basis for risk assessment of BPA.

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.

Biosynthesis of the Isocoumarin Derivatives Fusamarins is Mediated by the PKS8 Gene Cluster in Fusarium.

Fusarium mangiferae causes the mango malformation disease (MMD) on young mango trees and seedlings resulting in economically significant crop losses. In addition, F. mangiferae produces a vast array of secondary metabolites (SMs), including mycotoxins that may contaminate the harvest. Their production is tightly regulated at the transcriptional level. Here, we show that lack of the H3 K9-specific histone methyltransferase, FmKmt1, influences the expression of the F. mangiferae polyketide synthase (PKS) 8 (FmPKS8), a so far cryptic PKS. By a combination of reverse genetics, untargeted metabolomics, bioinformatics and chemical analyses including structural elucidation, we determined the FmPKS8 biosynthetic gene cluster (BGC) and linked its activity to the production of fusamarins (FMN), which can be structurally classified as dihydroisocoumarins. Functional characterization of the four FMN cluster genes shed light on the biosynthetic pathway. Cytotoxicity assays revealed moderate toxicities with IC50 values between 1 and 50 µM depending on the compound.

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.

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.

Detection of Mycotoxins in Highly Matrix-Loaded House-Dust Samples by QTOF-HRMS, IM-QTOF-HRMS, and TQMS: Advantages and Disadvantages.

The analysis of (trace) contaminants in environmental samples represents an important tool for exposure assessment and for the evaluation of potential risks to human health. Currently, mass spectrometric detection using triple quadrupole (TQMS) systems is the established method of choice. However, screening methods using high resolution mass spectrometry (HRMS) find increasing application as they provide advantages such as enhanced selectivity. A complex composition of environmental samples is known to have enormous effects on mass analyzers. The present work therefore compares the impact of a highly matrix-loaded sample material like house-dust on the performance of mass spectrometric detection of the emerging indoor contaminant group of mycotoxins by quadrupole time-of-flight (QTOF) and TQMS after ultrahigh-performance liquid chromatographic separation. Furthermore, the role of ionization efficiencies of different ion sources in instrument sensitivity was compared using an electrospray ionization source and a newly developed heated electrospray ion source (Bruker VIP-HESI) during QTOF experiments. Finally, it was evaluated whether an additional dimension of separation enables increased sensitivity in QTOF-HRMS detection by applying mycotoxins in house-dust to an (trapped) ion mobility spectrometry instrument. The sensitivity of the QTOF detection was positively influenced by the application of the VIP-HESI ion source, and overall HRMS instruments provided enhanced selectivity resulting in simplified data evaluation compared to the TQMS. However, all performed experiments revealed strong signal suppression due to matrix components. QTOF results showed more severe effects, enabling a more sensitive detection of mycotoxins in house-dust by applying TQMS detection.

Metabolic profiling as a powerful tool for the analysis of cellular alterations caused by 20 mycotoxins in HepG2 cells.

Mycotoxins are secondary fungal metabolites which exhibit toxic effects in low concentrations. Several mycotoxins are described as carcinogenic or immunosuppressive, but their underlying modes of action especially on molecular level have not yet been entirely elucidated. Metabolic profiling as part of the omics methods is a powerful tool to study the toxicity and the mode of action of xenobiotics. The use of hydrophilic interaction chromatography in combination with targeted mass spectrometric detection enables the selective and sensitive analysis of more than 100 polar and ionic metabolites and allows the evaluation of metabolic alterations caused by xenobiotics such as mycotoxins. For metabolic profiling, the hepato-cellular carcinoma cell line HepG2 was treated with sub-cytotoxic concentrations of 20 mycotoxins. Moniliformin and citrinin significantly affected target elements of the citric acid cycle, but also influenced glycolytic pathways and energy metabolism. Penitrem A, zearalenone, and T2 toxin mainly interfered with the urea cycle and the amino acid homeostasis. The formation of reactive oxygen species seemed to be influenced by T2 toxin and gliotoxin. Glycolysis was altered by ochratoxin A and DNA synthesis was affected by several mycotoxins. The observed effects were not limited to these metabolic reactions as the metabolic pathways are closely interrelated. In general, metabolic profiling proved to be a highly sensitive tool for hazard identification in comparison to single-target cytotoxicity assays as metabolic alterations were already observed at sub-toxic concentrations. Metabolic profiling could therefore be a powerful tool for the overall evaluation of the toxic properties of xenobiotics.

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.

Salivary nitrate/nitrite and acetaldehyde in humans: potential combination effects in the upper gastrointestinal tract and possible consequences for the in vivo formation of N-nitroso compounds-a hypothesis.

Subsequent to the dietary uptake of nitrate/nitrite in combination with acetaldehyde/ethanol, combination effects resulting from the sustained endogenous exposure to nitrite and acetaldehyde may be expected. This may imply locoregional effects in the upper gastrointestinal tract as well as systemic effects, such as a potential influence on endogenous formation of N-nitroso compounds (NOC). Salivary concentrations of the individual components nitrate and nitrite and acetaldehyde are known to rise after ingestion, absorption and systemic distribution, thereby reflecting their respective plasma kinetics and parallel secretion through the salivary glands as well as the microbial/enzymatic metabolism in the oral cavity. Salivary excretion may also occur with certain drug molecules and food constituents and their metabolites. Therefore, putative combination effects in the oral cavity and the upper digestive tract may occur, but this has remained largely unexplored up to now. In this Guest Editorial, published evidence on exposure levels and biokinetics of nitrate/nitrite/NOx, NOC and acetaldehyde in the organism is reviewed and knowledge gaps concerning combination effects are identified. Research is suggested to be initiated to study the related unresolved issues.

Insights into Ergochromes of the Plant Pathogen Claviceps purpurea.

Claviceps purpurea is an ergot fungus known for its neurotropic alkaloids, which have been identified as the main cause of ergotism, a livestock and human disease triggered by ergot consumption. Tetrahydroxanthone dimers, the so-called ergopigments, presumably also contribute to this toxic effect. Overexpression of the cluster-specific transcription factor responsible for the formation of these pigments in C. purpurea led to the isolation of three new metabolites (8-10). The new pigments were characterized utilizing HRMS, NMR techniques, and CD spectroscopy and shown to be xanthone dimers. Secalonic acid A and its 2,4'- and 4,4'-linked isomers were also isolated, and their absolute configuration was investigated. The contribution of secalonic acid A, its isomers, and new metabolites to the toxicity of C. purpurea was investigated in HepG2 and CCF-STTG1 cells. Along with cytotoxic properties, secalonic acid A was found to inhibit topoisomerase I and II activity.

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.