Hans-Ulrich Humpf

Dietary exposure to mycotoxins—including aflatoxins (AFs), ochratoxin A (OTA), and citrinin (CIT)—represents an important health risk, particularly in low-resource settings with widespread food contamination. This study investigates mycotoxin exposure among 719 participants (433 women and 286 children) of a cluster-randomized trial in rural Bangladesh, using residual blood samples collected during the 2019 endline survey. A total of 712 whole blood and 578 serum samples were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Serum aflatoxin B1 (AFB ₁ )-lysine adducts were quantified following enzymatic digestion and solid-phase extraction. To evaluate potential health risks, we estimated probable daily intake (PDI) from blood concentrations of frequently detected mycotoxins. All whole blood samples contained at least two mycotoxins, and 84% of participants were co-exposed to at least three mycotoxins. Biomarkers for the main blood-relevant mycotoxins were frequently detected, with OTA detected in 100% of whole blood samples and its isomer, 2′R-ochratoxin A (2′R-OTA), detected in 98%, CIT in 91%, and Enniatin B (EnB) in 92%, while AFB ₁ -lysine—a biomarker of chronic exposure to the potent carcinogen AFB ₁ —was detected in nearly one-quarter of women and 4% of children with serum samples. Beauvericin was detected in 6% of samples. The PDI of OTA exceeded health-based guidance values in 80% of women and 99% of children, while the PDI of CIT was of concern for 12% of women and 27% of children. These findings demonstrate widespread co-exposure to multiple mycotoxins, with OTA, CIT, and AFB ₁ posing notable risks, underscoring the urgent need for targeted interventions to protect vulnerable populations.

Deoxynivalenol (DON) is a prevalent trichothecene mycotoxin in cereals that poses food and feed safety risks while causing important economic losses. Microbial biotransformation offers a selective, mild strategy for DON detoxification. Here, we screened aerobic soil-derived bacterial communities from diverse agricultural environments, using DON as the sole carbon source for this mycotoxin depletion. More than half of the tested enrichment samples showed a reduced DON signal, as observed by HPLC-UV. To assess the biological relevance, culture extracts were tested for cytotoxicity in HepG2 cells. Z13, a soil sample that depleted DON but produced no other detectable metabolites, showed reduced cytotoxicity, comparable to the negative control. In contrast, samples that depleted DON but produced 3-keto-DON remained toxic. High-resolution LC-MS analysis indicated the formation of metabolites putatively identified as 3-keto-DON in enrichment cultures and 3-epi-DON in a Devosia strain culture. Community composition was profiled with 16S rRNA gene amplicon sequencing, which showed that Z13 presented a remarkable drop in diversity upon microbial cultivation, and included genera such as Devosia, Nocardioides, and Pseudomonas. Together, these results provide integrated chemical, biological, and ecological evidence for aerobic DON biotransformation in soil-derived communities, identify pathway products, and highlight practical constraints related to community dependence and storage sensitivity.

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea and is frequently used in food supplements. In recent years, numerous studies have highlighted the bioactivity of polyphenols beyond their established role as radical scavengers. However, EGCG is highly unstable in slightly basic solutions such as cell culture medium. It therefore remains unclear whether the biological effects attributed to EGCG are caused by the parent compound itself or by its oxidation products, including the dimers examined here. In this study, the effects of EGCG focusing on apoptosis induction and histone deacetylases (HDAC) were compared with those of its major oxidation products, theasinensin A (TSA), theasinensin D (TSD), and oolongtheanin digallate (OTDG), in the human hepatocellular carcinoma cell line HepG2. The induction of cellular pathways involved in apoptosis was investigated using several in vitro biochemical approaches. Transcriptional analysis of apoptosis-associated genes revealed distinct expression profiles, and caspase activities were differentially affected by the test compounds. HDAC activity in nuclear protein extracts was significantly reduced after incubation with the stabilized oxidation products, whereas no comparable HDAC-inhibitory effect was observed after direct incubation of HepG2 cells. Nevertheless, HDAC gene expression, particularly of class I isoforms, was modulated by the test compounds in the low micromolar range. These effects diminished at concentrations associated with the onset of apoptosis. Furthermore, untargeted proteomics identified ribosomal proteins as additional cellular targets. Overall, these findings help to clarify the contribution of abundant EGCG oxidation products to the antiproliferative and HDAC modulating effects commonly attributed to the parent compound under cell culture conditions, underscoring the importance of investigating these oxidation products.

Matrix effects (ME) during LC-ESI-MS analysis are a commonly acknowledged issue for a variety of matrices and analytes. Although sample preparation techniques are steadily evolving to reduce ME, the complexity and variability of the urine matrix remain a challenge, especially for multi-analyte methods. To investigate the extent of ME implications on method performance and quantification, we used stable isotope-labelled standards (SIL-IS) of 11 mycotoxins to evaluate the magnitude and variability of ME in urine samples from two cohorts: Bangladeshi adult women (n = 50) and Swedish children of both sexes (n = 340). Significant ME differences were observed between the two cohorts for eight of the 11 mycotoxins. Additionally, intra-cohort ME variability turned out to be very high with interquartile ranges (IQR) above 15% for 14 out of 22 analyte-cohort combinations. Maximum IQR values were observed for sterigmatocystin in the Bangladeshi cohort (318%), strongly impacting quantitative results obtained with matrix(-matched) calibration. Further experiments on a small German cohort of four subjects, each providing four to five urine samples, revealed high variability of ME within each individual. Factors influencing ME were investigated, showing little to no impact of sex and a moderate impact of age for some analytes in the Swedish cohort. Nonetheless, especially the more polar analytes, showing stronger signal suppression, demonstrated clear correlation of ME with density and creatinine concentration of the urine samples. As a result, urine samples with very high or low density or creatinine values require careful handling in regard to sensitivity or quantification errors when matrix(-matched) calibration without SIL-IS is applied.

Mycotoxins pose a major health risk for humans in many parts of the world. For exposure assessment human biomonitoring (HBM) based on the analysis of serum, plasma, whole blood, urine or breast milk samples is a powerful approach which has been extensively used. However, there are several major challenges and pitfalls to be considered to achieve reliable and standardized data which can be used for risk assessment. These challenges include sampling (spot urine, 24 h urine, plasma, serum etc.) and analytical issues (missing reference standards, matrix effects, protein adducts, recovery, limit of detection, stability during storage, etc.). Further critical aspects needed for biomarker interpretation and human health risk evaluation are a lack of data for several mycotoxins such as (a) health-based guidance values (b) long-term biomarkers, (c) reliable food intake data (d) toxicokinetic parameters in humans, for example clearance and transfer rates to urine or milk and (e) HBM guidance values. In this review, these issues will be discussed based on recent HBM studies and recommendations for future studies as well as research needs will be derived. Graphical abstract

Genetic information of living cells is encoded by the specific arrangement of nucleobases in deoxyribonucleic acid (DNA). Even minor changes or modifications of these nucleobases, favored by many reactive sites, can significantly affect correct replication and DNA integrity leading to severe toxic effects. Analyzing these DNA modifications is highly complex and often requires non-specific assays for DNA damage or ³² P-postlabelling. A major limitation of these methods is their inability to provide structural information, a gap that can be addressed by instrumental analytical techniques. An additional major challenge is the selection of an appropriate biological system capable of reliable DNA adduct formation in high yields, since most compounds require metabolic activation prior to reacting with DNA. Therefore, the aim of this study was to develop a fast and simple workflow for sensitive DNA adduct analysis. In addition to highlighting the main pitfalls in sample preparation, this publication focuses on the comparison of biological systems in terms of metabolic activity, using six well-known carcinogens from different chemical classes. The combination of HepG2 cells and liver S9 fractions demonstrated comparable or even superior capabilities to primary hepatocytes and enabled the detection of DNA adducts from aflatoxin B₁, benzo[a]pyrene, methyleugenol as well as α-asarone and β-asarone, particularly after metabolic activation by the aryl hydrocarbon receptor agonist β-naphthoflavone. Notably, the adduct formation of phenylpropanoids was documented for the first time in a non-transfected cancer cell line using high-performance liquid chromatography coupled to mass spectrometry. Therefore, the method can be used to detect previously unknown DNA adducts from diverse chemical classes and provides structural insights into the formed DNA adducts.