Fumonisins as a possible contributory risk factor for primary liver cancer: A 3-year study of corn harvested in Haimen, China, by HPLC and ELISA

Employing HPLC fluorometry, gas-liquid chromatography (GLC) and a novel enzymelinked immunosorbent assay (ELISA) based on a monoclonal antibody, 40 corn samples, each collected in 1993 from agricultural stocks for human consumption in Haimen (Jiangsu County) and Penlai (Shandong Province), high- and low-risk areas for primary liver cancer (PLC) in China, respectively, were analysed for fumonisins (FBs), aflatoxins (AFs) and trichothecenes. Levels and positive rates of FBs and deoxynivalenol (DON) were significantly higher in Haimen than in Penlai. ELISA of the 40 corn samples harvested in the two areas in 1994 revealed that FB contamination levels and rates in these areas were comparable to those observed in 1993 in Haimen. ELISA analysis of 1993 and 1994 products revealed a wide occurrence of AFB₁ but the positive rates as well as levels were not significantly different between these areas. ELISA of the same sample number of corn harvested in 1995 revealed that FB contamination in Haimen was significantly higher than in Penlai. These 3-yearly surveys of corn samples (240 in total) demonstrated that corn harvested in Haimen was highly contaminated with FBs and that the contamination level, as well as positive rate in 1993 and 1995, were 10–50-fold higher than those in Penlai, suggesting FBs as a risk factor for promotion of PLC in endemic areas, along with the trichothecene DON. Co-contamination with AFs, potent hepatocarcinogens, was assumed to play an important role in the initiation of hepatocarcinogenesis.     

Modulation of murine host response to enteric reovirus infection by the trichothecene deoxynivalenol.

Based on the known capacity of deoxynivalenol (DON) to target gut lymphoid tissue and IgA production, it was hypothesized that this mycotoxin interferes with the immune response to enteric reovirus infection. When mice were orally gavaged, first with 25 mg/kg bw DON, and then with reovirus serotype 1, strain Lang (T1/L) 2 or 12 h later, viral titers in the GI tract were 10-fold higher than control mice after 5 days. Virus was almost completely cleared in both treatment and control groups from intestinal tissue after 10 days. Real-time PCR indicated that, in infected control mice, reovirus lambda2 core spike (L2 gene) RNA per g feces in infected mice that were pretreated with DON was significantly higher at 1, 3, and 5 days than in infected mice only. In reovirus-infected mice, DON at doses of 10 and 25 mg/kg bw but not 2 and 5 mg/kg bw increased fecal L2 RNA, whereas DON doses as low as 2 mg/kg potentiated L2 RNA levels in Peyer's patches (PP). Reovirus-specific IgA levels in feces of mice treated with DON were significantly elevated, as were specific IgA responses in lamina propria and PP fragment cultures. Similar effects were observed for serum IgA and IgG. DON suppressed IFN-gamma responses in PP to reovirus at 3 and 5 days as compared to infected controls, while IL-2 mRNA concentrations were unaffected. Although reovirus alone did not induce Th2 cytokine mRNAs in PP, DON exposure significantly elevated IL-4, IL-6, and IL-10 mRNA expression at various times during the infection. ELISPOT revealed that mRNA expression data corresponded to suppression of IFN-gamma- and enhancement of IL-4-producing cell responses in PP cultures from DON-treated mice. Taken together, these data suggest that DON transiently increased both severity of the reovirus infection and shedding in feces as well as elevated reovirus IgA responses. These effects corresponded to suppressed Th1 and enhanced Th2 cytokine expression.     

Mycotoxin Exposure in Children through Breakfast Cereal Consumption in Chile

Mycotoxins are unavoidable contaminants produced by fungi in food, especially grains. This study aimed to measure the occurrence and levels of total aflatoxins (AFs); ochratoxin A (OTA); zearalenone (ZEN); fumonisins B1, B2, and B3 (FUM); deoxynivalenol (DON); and T-2/HT-2 toxins in the four most commonly consumed breakfast cereals in Chile and to assess mycotoxin exposure and risk in children aged 2 to 13 years due to cereal consumption. In this study, a total of 110 batches with three subsamples of the four brands were sampled in supermarkets from November 2019 to June 2021. Samples were analyzed by Veratox^® ELISA (Neogen). Exposure was assessed by estimated daily intake (EDI) considering the levels found in a modified lower bound (mLB) and upper bound (UB). Risk was estimated by margin of exposure (MOE) in the case of OTA and AFs and hazard quotient (HQ) for the rest of the mycotoxins. No T2/HT2 toxins were detected. Few samples had quantifiable levels of ZEN, FUM, and DON except for brand 1, with a mean (standard deviation, SD) of 54 (20), 1552 (351), and 706 (218) ng/g, respectively. In addition, three FUM samples and one DON sample had values over the Chilean regulation. Brands 2, 3, and 4 had quantifiable levels of AFs, with mean (SD) values of 1.3 (0.1), 2.1 (0.6), and 1.9 (0.4) ng/g, respectively. Brand 3 had quantifiable levels of OTA, with a mean (SD) of 2.3 (0.4) ng/g. Estimated exposure indicated a risk of AFs in all scenarios, and of FUM for brand 1 consumption, OTA and DON for brand 3 consumption, and OTA for brand 4 consumption in the mLB worst-case scenario. In general, mycotoxin levels were below the Chilean regulatory limits, but most of them were above the EU regulation for processed cereal-based food in young children. Because the risk was higher in the 2- to 5-year-old children, we recommend special regulations for this group in Chile.     

The Role of Nitrogen Fertilization on the Occurrence of Regulated,Modified and Emerging Mycotoxins and Fungal Metabolites in Maize Kernels

The European Food Safety Authority is currently evaluating the risks related to the presence of emerging mycotoxins in food and feeds. The aim of this study was to investigate the role of soil fertility, resulting from different nitrogen fertilization rates, on the contamination of regulated mycotoxins and emerging fungal metabolites in maize grains. The trial was carried out in the 2012–2013 growing seasons as part of a long-term (20-year) experimental platform area in North-West Italy, where five different N rates, ranging from 0 to 400 kg N ha⁻¹, were applied to maize each year. Maize samples were analyzed by means of a dilute-and-shoot multi-mycotoxin LC-MS/MS method, and more than 25 of the most abundant mycotoxins and fungal metabolites were detected. Contamination by fumonisins and other fungal metabolites produced by Fusarium spp. of the section Liseola was observed to have increased in soils that showed a poor fertility status. On the other hand, an overload of nitrogen fertilization was generally associated with higher deoxynivalenol and zearalenone contamination in maize kernels, as well as a higher risk of other fungal metabolites produced by Fusarium spp. sections Discolor and Roseum. A balanced application of N fertilizer, in accordance with maize uptake, generally appears to be the best solution to guarantee an overall lower contamination by regulated mycotoxins and emerging fungal metabolites.     

Deterministic and Probabilistic Dietary Exposure Assessment to Deoxynivalenol in Spain and the Catalonia Region

Deoxynivalenol (DON) remains one of the most concerning mycotoxins produced by the Fusarium genus due to the wide occurrence in highly consumed cereal-based food and its associated toxicological effects. Previous studies conducted in Spain and other European countries suggested that some vulnerable groups such as children could be exceeding the tolerable daily intakes. Thus, the aim of this study was to conduct a comprehensive and updated dietary exposure assessment study in Spain, with a specific analysis in the region of Catalonia. Cereal-based food samples collected during 2019 were analysed using liquid chromatography coupled to tandem mass spectrometry for multi-mycotoxin detection including DON and its main metabolites and derivatives. Consumption data were gathered from the nation-wide food surveys ENALIA and ENALIA2 conducted in Spain, and a specific survey conducted in Catalonia. The data were combined using deterministic and semi-parametric probabilistic methods. The results showed that DON was widely present in cereal-based food highly consumed in Spain and the Catalonia region. Exposure to DON among the adult population was globally low; however, among infants aged 3–9 years, it resulted in the median of 192 ng/kg body weight/day and the 95th percentiles of 604 ng/kg body weight/day, that would exceed the most conservative safety threshold for infants. Bread and pasta were the main contributing foodstuffs to the global exposure to DON, even among infants; thus, those foods should be considered a priority for food control or to develop strategies to reduce the exposure. In any case, further toxicological and epidemiological studies are required in order to refine the safety thresholds accounting for the sensitivity of the infant population.     

一种酶联免疫检测系统用于米面中真菌毒素检测的效果评价

目的：评价某品牌数字化食品安全快速检测系统对米面中黄曲霉毒素B1（AFB1）、脱氧雪腐镰刀菌烯醇（DON）及玉米赤霉烯酮（ZEN）3种真菌毒素的酶联免疫检测效果。方法：以从超市随机购买的某品牌大米和面粉作为对象,按照试剂盒说明书开展酶联免疫吸附测定（ELISA）检测,并进行标准曲线线性、各种毒素最低检测限、加标回收率及加标回收精密度等方法学验证。结果：3种毒素标准曲线的线性相关系数均大于0.99;AFB1、DON及ZEN的最低检测限分别为1.69、184及41μg/kg,均低于食品中真菌毒素限量国家标准;平均加标回收率在80%~100%之间,加标回收的相对标准偏差（RSD）在10.0以内。结论：该品牌数字化食品安全快速检测系统及配套试剂盒对米面中真菌毒素检测的样品前处理简单、检测速度快、结果准确性高,具有较强的适用性。     

Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line

Abstract

Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3?h to 0.5?µM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF-κB (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit?→?ribotoxic stress?→?ER stress?→?calcium release from the ER into cytoplasm?→?T-cell activation and oxidative stress?→?apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell activation response by increased intracellular calcium levels.     

T-2毒素、脱氧雪腐镰刀菌烯醇全抗原的合成及鉴定

目的分别合成T-2毒素、脱氧雪腐镰刀菌烯醇(DON)的全抗原。方法采用琥珀酸酐法对分子结构进行修饰,产生易反应的官能团,并与载体蛋白偶联,从而具备免疫条件,用高性能基质辅助激光解吸电离-飞行时间质谱仪MALDI-TOF检测偶联物及偶联蛋白的分子量,用商品化的T-2和DON免疫试剂盒与偶联物进行棋盘滴定验证。结果T-2和DON的全抗原及BSA的分子量分别为67641.6、66680.4、66297.7,偶联物与免疫试剂盒中T-2和DON的抗体均呈阳性反应。结论合成的T-2、DON全抗原为相应抗体的制备和免疫学方法的建立奠定了基础。     

Toxicology of 3-epi-deoxynivalenol,a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8

Microbial detoxification of deoxynivalenol (DON) represents a new approach to treating DON-contaminated grains. A bacterium Devosia mutans 17-2-E-8 was capable of completely transforming DON into a major product 3-epi-DON and a minor product 3-keto-DON. Evaluation of toxicities of these DON-transformation products is an important part of hazard characterization prior to commercialization of the biotransformation application. Cytotoxicities of the products were demonstrated by two assays: a MTT bioassay assessing cell viability and a BrdU assay assessing DNA synthesis. Compared with DON, the IC₅₀ values of 3-epi-DON and 3-keto-DON were respectively 357 and 3.03 times higher in the MTT bioassay, and were respectively 1181 and 4.54 times higher in the BrdU bioassay. Toxicological effects of 14-day oral exposure of the B6C3F₁ mouse to DON and 3-epi-DON were also investigated. Overall, there were no differences between the control (free of toxin) and the 25 mg/kg bw/day or 100 mg/kg bw/day 3-epi-DON treatments in body and organ weights, hematology and organ histopathology. However, in mice exposed to DON (2 mg/kg bw/day), white blood cell numbers and serum immunoglobulin levels were altered relative to controls, and lesions were observed in adrenals, thymus, stomach, spleen and colon. Taken together, in vitro and in vivo studies indicate that 3-epi-DON is substantially less toxic than DON.