2024

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12 pages, 1947 KiB

Open AccessArticle

Inhibition of Aflatoxin Production in Aspergillus flavus by a Klebsiella sp. and Its Metabolite Cyclo(l-Ala-Gly)

by Shohei Sakuda, Masaki Sunaoka, Maho Terada, Ayaka Sakoda, Natsumi Ishijima, Noriko Hakoshima, Kenichi Uchida, Hirofumi Enomoto and Tomohiro Furukawa

Toxins 2024, 16(3), 141; https://doi.org/10.3390/toxins16030141 - 08 Mar 2024

Viewed by 814

Abstract

During an experiment where we were cultivating aflatoxigenic Aspergillus flavus on peanuts, we accidentally discovered that a bacterium adhering to the peanut strongly inhibited aflatoxin (AF) production by A. flavus. The bacterium, isolated and identified as Klebsiella aerogenes, was found to [...] Read more.

During an experiment where we were cultivating aflatoxigenic Aspergillus flavus on peanuts, we accidentally discovered that a bacterium adhering to the peanut strongly inhibited aflatoxin (AF) production by A. flavus. The bacterium, isolated and identified as Klebsiella aerogenes, was found to produce an AF production inhibitor. Cyclo(l-Ala-Gly), isolated from the bacterial culture supernatant, was the main active component. The aflatoxin production-inhibitory activity of cyclo(l-Ala-Gly) has not been reported. Cyclo(l-Ala-Gly) inhibited AF production in A. flavus without affecting its fungal growth in a liquid medium with stronger potency than cyclo(l-Ala-l-Pro). Cyclo(l-Ala-Gly) has the strongest AF production-inhibitory activity among known AF production-inhibitory diketopiperazines. Related compounds in which the methyl moiety in cyclo(l-Ala-Gly) is replaced by ethyl, propyl, or isopropyl have shown much stronger activity than cyclo(l-Ala-Gly). Cyclo(l-Ala-Gly) did not inhibit recombinant glutathione-S-transferase (GST) in A. flavus, unlike (l-Ala-l-Pro), which showed that the inhibition of GST was not responsible for the AF production-inhibition of cyclo(l-Ala-Gly). When A. flavus was cultured on peanuts dipped for a short period of time in a dilution series bacterial culture broth, AF production in the peanuts was strongly inhibited, even at a 1 × 10⁴-fold dilution. This strong inhibitory activity suggests that the bacterium is a candidate for an effective biocontrol agent for AF control. Full article

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2023

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12 pages, 294 KiB

Open AccessArticle

Assessment of the Adverse Health Effects of Aflatoxin Exposure from Unpackaged Peanut Oil in Guangdong, China

by Zhini He, Zihui Chen, Yunying Mo, Xiaodan Lu, Yanheng Luo, Shaoliang Lin, Yanxu Zhong, Junfeng Deng, Shixiong Zheng, Lei Xia, Hang Wu, Michael N. Routledge, Ye Hong, Xiaoyu Xian, Xingfen Yang and Yunyun Gong

Toxins 2023, 15(11), 646; https://doi.org/10.3390/toxins15110646 - 09 Nov 2023

Viewed by 1651

Abstract

Aflatoxins are liver carcinogens and are common contaminants in unpackaged peanut (UPP) oil. However, the health risks associated with consuming aflatoxins in UPP oil remain unclear. In this study, aflatoxin contamination in 143 UPP oil samples from Guangdong Province were assessed via liquid [...] Read more.

Aflatoxins are liver carcinogens and are common contaminants in unpackaged peanut (UPP) oil. However, the health risks associated with consuming aflatoxins in UPP oil remain unclear. In this study, aflatoxin contamination in 143 UPP oil samples from Guangdong Province were assessed via liquid chromatography–tandem mass spectrometry (LC-MS). We also recruited 168 human subjects, who consumed this oil, to measure their liver functions and lipid metabolism status. Aflatoxin B1 (AFB1) was detected in 79.72% of the UPP oil samples, with levels ranging from 0.02 to 174.13 μg/kg. The average daily human intake of AFB1 from UPP oil was 3.14 ng/kg·bw/day; therefore, the incidence of liver cancer, caused by intake of 1 ng/kg·bw/day AFB1, was estimated to be 5.32 cases out of every 100,000 persons per year. Meanwhile, Hepatitis B virus (HBV) infection and AFB1 exposure exerted a synergistic effect to cause liver dysfunction. In addition, the triglycerides (TG) abnormal rate was statistically significant when using AFB1 to estimate daily intake (EDI) quartile spacing grouping (p = 0.011). In conclusion, high aflatoxin exposure may exacerbate the harmful effects of HBV infection on liver function. Contamination of UPP oil with aflatoxins in Guangdong urgently requires more attention, and public health management of the consumer population is urgently required. Full article

16 pages, 6000 KiB

Open AccessArticle

Isolation and Optimization of Aflatoxin B₁ Degradation by Uniform Design and Complete Genome Sequencing of Novel Deep-Sea Kocuria rosea Strain 13

by Jingying Wang, Qiqi Chen, Peisheng Yan, Chunming Dong and Zongze Shao

Toxins 2023, 15(9), 520; https://doi.org/10.3390/toxins15090520 - 24 Aug 2023

Viewed by 957

Abstract

Aflatoxin B₁ is a natural carcinogenic mycotoxin. The biological detoxification of aflatoxin could result in less environmental pollution, more moderate conditions, and less impact on food and feed, and be more convenient than physical and chemical methods. In this study, strain 13 [...] Read more.

Aflatoxin B₁ is a natural carcinogenic mycotoxin. The biological detoxification of aflatoxin could result in less environmental pollution, more moderate conditions, and less impact on food and feed, and be more convenient than physical and chemical methods. In this study, strain 13 with aflatoxin B₁ degradation activity (67.47 ± 1.44%) was isolated and identified as Kocuria rosea. A uniform design was applied to optimize the degradation activity using a software Data Processing System, and a quadratic polynomial stepwise regression model was selected to investigate the relationships between the degradation rate and five independent variables. Furthermore, the optimal degradation conditions (culture temperature of 30 °C, culture time of 4.2 days, seawater ratio of 100%, pH of 7.11, and inoculation dosage of 0.09%) were verified with a degradation rate of 88 ± 0.03%, which was well matched with the predicted value (92.97%) of the model. Complete genome sequencing of Kocuria rosea, conducted with a combination of Illumina and single-molecule real-time sequencing, was used to analyze the genomic features and functions of the strain, which were predicted by the annotation based on seven databases, and may provide insights into the potential of Kocuria rosea, as well as providing a reference for degradation gene and protein mining. These results indicate that Kocuria rosea strain 13 has the ability to degrade aflatoxin B₁ efficiently, and it also has the potential to provide aflatoxin-degrading enzymes. Full article

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16 pages, 3054 KiB

Open AccessArticle

A Novel Multicellular Placental Barrier Model to Investigate the Effect of Maternal Aflatoxin B₁ Exposure on Fetal-Side Neural Stem Cells

by Zhiwei Zhou, Dongmei Luo, Mengxue Li, Guangjie Lao, Zhiqiang Zhou, András Dinnyés, Wenming Xu and Qun Sun

Toxins 2023, 15(5), 312; https://doi.org/10.3390/toxins15050312 - 27 Apr 2023

Cited by 2 | Viewed by 1966

Abstract

Ingestion of food toxins such as aflatoxin B₁ (AFB₁) during pregnancy may impair fetal neurodevelopment. However, animal model results may not be accurate due to the species’ differences, and testing on humans is ethically impermissible. Here, we developed an in [...] Read more.

Ingestion of food toxins such as aflatoxin B₁ (AFB₁) during pregnancy may impair fetal neurodevelopment. However, animal model results may not be accurate due to the species’ differences, and testing on humans is ethically impermissible. Here, we developed an in vitro human maternal–fetal multicellular model composed of a human hepatic compartment, a bilayer placental barrier, and a human fetal central nervous system compartment using neural stem cells (NSCs) to investigate the effect of AFB₁ on fetal-side NSCs. AFB₁ passed through the HepG2 hepatocellular carcinoma cells to mimic the maternal metabolic effects. Importantly, even at the limited concentration (0.0641 ± 0.0046 μM) of AFB_1, close to the national safety level standard of China (GB-2761-2011), the mixture of AFB₁ crossing the placental barrier induced NSC apoptosis. The level of reactive oxygen species in NSCs was significantly elevated and the cell membrane was damaged, causing the release of intracellular lactate dehydrogenase (p < 0.05). The comet experiment and γ-H2AX immunofluorescence assay showed that AFB₁ caused significant DNA damage to NSCs (p < 0.05). This study provided a new model for the toxicological evaluation of the effect of food mycotoxin exposure during pregnancy on fetal neurodevelopment. Full article

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13 pages, 1822 KiB

Open AccessEditor’s ChoiceArticle

Dispersive Magnetic Solid-Phase Extraction as a Novelty Sample Treatment for the Determination of the Main Aflatoxins in Paprika

by María García-Nicolás, Natalia Arroyo-Manzanares and Pilar Viñas

Toxins 2023, 15(2), 160; https://doi.org/10.3390/toxins15020160 - 15 Feb 2023

Cited by 3 | Viewed by 1917

Abstract

Dispersive magnetic solid-phase extraction (DMSPE) technique is proposed as a new sensitive and effective sample treatment method for the determination of aflatoxins in paprika samples. DMSPE was followed by ultrahigh-performance liquid chromatography and high-resolution mass spectrometry detection (UHPLC-HRMS) using a non-targeted acquisition mode [...] Read more.

Dispersive magnetic solid-phase extraction (DMSPE) technique is proposed as a new sensitive and effective sample treatment method for the determination of aflatoxins in paprika samples. DMSPE was followed by ultrahigh-performance liquid chromatography and high-resolution mass spectrometry detection (UHPLC-HRMS) using a non-targeted acquisition mode for the detection of main aflatoxins (aflatoxin G1, G2, B1 and B2) and derivatives. DMSPE was based on the use of magnetic nanocomposite coated with polypyrrole (PPy) polymer and the main experimental parameters influencing the extraction efficiency in adsorption and desorption steps have been studied and optimized. Analyses were performed using 250 µL magnetic PPy nanocomposite into the sample solution, adsorbing the analytes in 30 min and desorbing them with ethyl acetate (2 mL) in 15 min. The method has been validated, obtaining quantification limits between 3.5 and 4.7 µg kg⁻¹ and recoveries between 89.5–97.7%. The high recovery rate, wide detection range and the use for the first time of the reusable Fe₃O₄@PPy nanomaterial in suspension for solid food matrices, guarantee the usefulness of the method developed for adequate control of aflatoxins levels in paprika. The proposed methodology was applied for the analysis of 31 samples (conventional and organic) revealing the absence of aflatoxins in the samples. Full article

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13 pages, 431 KiB

Open AccessEditor’s ChoiceArticle

The Effects of Incremental Doses of Aflatoxin B₁ on In Vitro Ruminal Nutrient Digestibility and Fermentation Profile of a Lactating Dairy Cow Diet in a Dual-Flow Continuous Culture System

by Felipe Xavier Amaro, Yun Jiang, Kathy Arriola, Matheus R. Pupo, Bruna C. Agustinho, Sarah L. Bennett, James R. Vinyard, Lais Tomaz, Richard R. Lobo, Andres Pech-Cervantes, Jose A. Arce-Cordero, Antonio P. Faciola, Adegbola Tolulope Adesogan and Diwakar Vyas

Toxins 2023, 15(2), 90; https://doi.org/10.3390/toxins15020090 - 18 Jan 2023

Cited by 1 | Viewed by 1850

Abstract

Aflatoxin B₁ (AFB₁) is a mycotoxin known to impair human and animal health. It is also believed to have a deleterious effect on ruminal nutrient digestibility under in vitro batch culture systems. The objective of this study was to evaluate [...] Read more.

Aflatoxin B₁ (AFB₁) is a mycotoxin known to impair human and animal health. It is also believed to have a deleterious effect on ruminal nutrient digestibility under in vitro batch culture systems. The objective of this study was to evaluate the effects of increasing the dose of AFB₁ on ruminal dry matter and nutrient digestibility, fermentation profile, and N flows using a dual-flow continuous culture system fed a diet formulated for lactating dairy cows. Eight fermenter vessels were used in a replicated 4 × 4 Latin square design with 10 d periods (7 d adaptation and 3 d sample collection). Treatments were randomly applied to fermenters on diet DM basis: (1) 0 μg of AFB₁/kg of DM (Control); (2) 50 μg of AFB₁/kg of DM (AF50); (3) 100 μg of AFB₁/kg of DM (AF100); and (4) 150 μg of AFB₁/kg of DM (AF150). Treatments did not affect nutrient digestibility, fermentation, and N flows. Aflatoxin B₁ concentration in ruminal fluid increased with dose but decreased to undetectable levels after 4 h post-dosing. In conclusion, adding incremental doses of AFB₁ did not affect ruminal fermentation, digestibility of nutrients, and N flows in a dual-flow continuous culture system fed diets formulated for lactating dairy cows. Full article

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2022

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12 pages, 499 KiB

Open AccessArticle

Chronic Aflatoxin Exposure and Cognitive and Language Development in Young Children of Bangladesh: A Longitudinal Study

by Mustafa Mahfuz, Md. Shabab Hossain, Md. Ashraful Alam, Md. Amran Gazi, Shah Mohammad Fahim, Baitun Nahar and Tahmeed Ahmed

Toxins 2022, 14(12), 855; https://doi.org/10.3390/toxins14120855 - 03 Dec 2022

Cited by 4 | Viewed by 1651

Abstract

Aflatoxin can cross the blood–brain barrier, damage brain tissues, and have the potential to harm the development of the human brain. Although dietary aflatoxin exposure is common in children, there is a paucity of data on aflatoxin exposure and child developmental outcomes. The [...] Read more.

Aflatoxin can cross the blood–brain barrier, damage brain tissues, and have the potential to harm the development of the human brain. Although dietary aflatoxin exposure is common in children, there is a paucity of data on aflatoxin exposure and child developmental outcomes. The child’s cognitive, motor, and language functions were assessed using the Bayley Scales of Infant and Toddler Development-III or BSID-III at the same time points. Association between exposure to aflatoxin and subtests of BSID-III were examined using mixed-effect linear regression. Aflatoxin assays were performed on 194, 167, and 163 children at 15, 24, and 36 months of age, and chronic aflatoxin exposure was detected in 20.6%, 16.8%, and 60.7% of children, respectively. Multi-variable analyses showed that aflatoxin exposure was independently related to the children’s cognitive score (β: −0.69; 95% CI: −1.36, −0.02), receptive language score (β: −0.90; 95% CI: −1.62, −0.17), and expressive language score (β: −1.01; 95% CI: −1.96, −0.05). We did not observe any association between exposure to aflatoxin and the motor function of children. Chronic exposure to aflatoxin exposure was linked to reduced cognitive, expressive, and receptive language scores of the study children. Further research is needed in a different setting to confirm this novel finding. Full article

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11 pages, 1409 KiB

Open AccessArticle

Effects of Four Isothiocyanates in Dissolved and Gaseous States on the Growth and Aflatoxin Production of Aspergillus flavus In Vitro

by Yohei Hareyama, Mitsunori Tarao, Koki Toyota, Tomohiro Furukawa, Yoshiharu Fujii and Masayo Kushiro

Toxins 2022, 14(11), 756; https://doi.org/10.3390/toxins14110756 - 02 Nov 2022

Cited by 3 | Viewed by 1347

Abstract

Aflatoxins (AFs), a class of toxins produced by certain species of the genus Aspergillus, occasionally contaminate food and cause serious damage to human health and the economy. AFs contamination is a global problem, and there is a need to develop effective strategies [...] Read more.

Aflatoxins (AFs), a class of toxins produced by certain species of the genus Aspergillus, occasionally contaminate food and cause serious damage to human health and the economy. AFs contamination is a global problem, and there is a need to develop effective strategies to control aflatoxigenic fungi. In this study, we focused on isothiocyanates (ITCs) as potential chemical agents for the control of aflatoxigenic fungi. We quantitatively evaluated the effects of four ITCs (allyl ITC (AITC), benzyl ITC (BITC), and methyl and phenylethyl ITCs) in dissolved and gaseous states on the growth and aflatoxin B₁ production of Aspergillus flavus. In experiments using dissolved ITCs, BITC was found to be the strongest inhibitor of growth and aflatoxin B₁ production by A. flavus. Meanwhile, in the gaseous state, AITC strongly inhibited the A. flavus growth. When the concentration of ITCs in the liquid medium was quantified over time, AITC levels decreased to below the detection limit within 24 h, whereas BITC levels remained stable even after 48 h. These results suggested that when ITCs are utilized to control aflatoxigenic fungi, it is necessary to use them in a dissolved or gaseous state, depending on their volatility. Full article

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20 pages, 5838 KiB

Open AccessArticle

Evaluation of Toxicant-Associated Fatty Liver Disease and Liver Neoplastic Progress in Sprague-Dawley Rats Treated with Low Doses of Aflatoxin B1 Alone or in Combination with Extremely Low Frequency Electromagnetic Fields

by Andrea Vornoli, Eva Tibaldi, Federica Gnudi, Daria Sgargi, Fabiana Manservisi, Fiorella Belpoggi, Francesco Tovoli and Daniele Mandrioli

Toxins 2022, 14(5), 325; https://doi.org/10.3390/toxins14050325 - 03 May 2022

Cited by 3 | Viewed by 2362

Abstract

The term toxicant-associated fatty liver disease (TAFLD) has been proposed to describe fatty liver diseases connected to toxicants other than alcohol. Aflatoxins are mycotoxins commonly found as contaminants in foods and feeds, which are known liver toxicants and potential candidates as potential causes [...] Read more.

The term toxicant-associated fatty liver disease (TAFLD) has been proposed to describe fatty liver diseases connected to toxicants other than alcohol. Aflatoxins are mycotoxins commonly found as contaminants in foods and feeds, which are known liver toxicants and potential candidates as potential causes of TAFLD. Aflatoxin B1 (AFB1) was administered at low doses to Sprague-Dawley (SD) rats, alone or in combination with S-50 Hz an extremely low frequency electromagnetic field (ELFEMF), to study the evolution of TAFLD, preneoplastic and neoplastic lesions of the liver and the potential enhancing effect of lifespan exposure to ELFEMF. Steatosis, inflammation and foci of different types were significantly increased in both aflatoxin-treated males and females, which is consistent with a pattern of TAFLD. A significant increase in adenomas, cystic dilation of biliary ducts, hepatocellular hyperplasia and hypertrophy and oval cell hyperplasia were also observed in treated females only. The administration of low doses of AFB1 caused TAFLD in SD rats, inducing liver lesions encompassing fatty infiltration, foci of different types and adenomas. Furthermore, the pattern of change observed in preneoplastic liver lesions often included liver steatosis and steatohepatitis (TASH). ELFEMF did not result in any enhancing or toxic effect in the liver of SD rats. Full article

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16 pages, 1884 KiB

Open AccessArticle

Comparison Evaluation of the Biological Effects of Sterigmatocystin and Aflatoxin B1 Utilizing SOS-Chromotest and a Novel Zebrafish (Danio rerio) Embryo Microinjection Method

by Zsolt Csenki, Anita Risa, Dorottya Sárkány, Edina Garai, Ildikó Bata-Vidács, Erzsébet Baka, András Szekeres, Mónika Varga, András Ács, Jeffrey Griffitts, Katalin Bakos, Illés Bock, István Szabó, Balázs Kriszt, Béla Urbányi and József Kukolya

Toxins 2022, 14(4), 252; https://doi.org/10.3390/toxins14040252 - 31 Mar 2022

Cited by 2 | Viewed by 2549

Abstract

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC [...] Read more.

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment. Full article

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23 pages, 395 KiB

Open AccessArticle

Effect of Dietary L-Threonine and Toxin Binder on Performance, Blood Parameters, and Immune Response of Broilers Exposed to Aflatoxin B₁

by Aydin Mesgar, Habib Aghdam Shahryar, Christopher Anthony Bailey, Yahya Ebrahimnezhad and Anand Mohan

Toxins 2022, 14(3), 192; https://doi.org/10.3390/toxins14030192 - 04 Mar 2022

Cited by 6 | Viewed by 3070

Abstract

To evaluate the effect of L-Threonine (L-Thr) and Mycofix^® Plus (MP) on aflatoxicosis, an experiment with a 3-way ANOVA model was carried out with 8 replicates and 640 birds. Treatments included two levels of L-Thr (100% and 125% of the requirements, Cobb [...] Read more.

To evaluate the effect of L-Threonine (L-Thr) and Mycofix^® Plus (MP) on aflatoxicosis, an experiment with a 3-way ANOVA model was carried out with 8 replicates and 640 birds. Treatments included two levels of L-Thr (100% and 125% of the requirements, Cobb 500, Cobb-Vantress), Aflatoxin B₁ (AFB₁) (0, 500 ppb), and MP (0, 1 g/kg). As the main effects showed, AFB₁ decreased breast meat yield and carcass percentage (p < 0.001), serum urea, antibody titer against infectious bronchitis virus (IBV), and bone density (p < 0.05), while it increased the plasma concentrations of glucose and alkaline phosphatase (ALP) (p < 0.05). Mycofix Plus improved the grower feed intake (FI), tibia fresh weight, and body weight (BW) to bone weight (p < 0.05). L-Threonine increased the grower FI, breast meat yield, serum aspartate transaminase (AST), and glutathione peroxidase (GPX) (p < 0.05). There were positive interactions with breast meat yield, cholesterol, lactate dehydrogenase (LDH), and IBV titer. Of the treatments used, the combination of L-Thr and MP without AFB₁ improved breast meat and carcass percentage. L-Threonine and MP significantly improved IBV titer in birds challenged with AFB₁ (p < 0.001). In conclusion, L-Thr and MP were beneficial to improve immunity. Full article

2021

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14 pages, 2976 KiB

Open AccessArticle

EGCG Alleviates Oxidative Stress and Inhibits Aflatoxin B₁ Biosynthesis via MAPK Signaling Pathway

by Dan Xu, Shurui Peng, Rui Guo, Lishan Yao, Haizhen Mo, Hongbo Li, Hongxin Song and Liangbin Hu

Toxins 2021, 13(10), 693; https://doi.org/10.3390/toxins13100693 - 30 Sep 2021

Cited by 17 | Viewed by 2593

Abstract

Aflatoxin biosynthesis has established a connection with oxidative stress, suggesting a prevention strategy for aflatoxin contamination via reactive oxygen species (ROS) removal. Epigallocatechin gallate (EGCG) is one of the most active and the richest molecules in green tea with well-known antioxidant effects. Here, [...] Read more.

Aflatoxin biosynthesis has established a connection with oxidative stress, suggesting a prevention strategy for aflatoxin contamination via reactive oxygen species (ROS) removal. Epigallocatechin gallate (EGCG) is one of the most active and the richest molecules in green tea with well-known antioxidant effects. Here, we found EGCG could inhibit aflatoxin B₁ (AFB₁) biosynthesis without affecting mycelial growth in Aspergillus flavus, and the arrest occurred before the synthesis of toxin intermediate metabolites. Further RNA-seq analysis indicated that multiple genes involved in AFB₁ biosynthesis were down-regulated. In addition, EGCG exposure facilitated the significantly decreased expression of AtfA which is a bZIP (basic leucine zipper) transcription factor mediating oxidative stress. Notably, KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis indicated that the MAPK signaling pathway target transcription factor was down-regulated by 1 mg/mL EGCG. Further Western blot analysis showed 1 mg/mL EGCG could decrease the levels of phosphorylated SakA in both the cytoplasm and nucleus. Taken together, these data evidently supported that EGCG inhibited AFB₁ biosynthesis and alleviated oxidative stress via MAPK signaling pathway. Finally, we evaluated AFB₁ contamination in soy sauce fermentation and found that EGCG could completely control AFB₁ contamination at 8 mg/mL. Conclusively, our results supported the potential use of EGCG as a natural agent to prevent AFB₁ contamination in fermentation industry. Full article

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20 pages, 2496 KiB

Open AccessEditor’s ChoiceArticle

Saccharomyces cerevisiae Cell Wall-Based Adsorbent Reduces Aflatoxin B1 Absorption in Rats

by Alexandros Yiannikouris, Juha Apajalahti, Osmo Siikanen, Gerald Patrick Dillon and Colm Anthony Moran

Toxins 2021, 13(3), 209; https://doi.org/10.3390/toxins13030209 - 13 Mar 2021

Cited by 17 | Viewed by 3377

Abstract

Mycotoxins are naturally occurring toxins that can affect livestock health and performance upon consumption of contaminated feedstuffs. To mitigate the negative effects of mycotoxins, sequestering agents, adsorbents, or binders can be included to feed to interact with toxins, aiding their passage through the [...] Read more.

Mycotoxins are naturally occurring toxins that can affect livestock health and performance upon consumption of contaminated feedstuffs. To mitigate the negative effects of mycotoxins, sequestering agents, adsorbents, or binders can be included to feed to interact with toxins, aiding their passage through the gastrointestinal tract (GI) and reducing their bioavailability. The parietal cell wall components of Saccharomyces cerevisiae have been found to interact in vitro with mycotoxins, such as, but not limited to, aflatoxin B1 (AFB1), and to improve animal performance when added to contaminated diets in vivo. The present study aimed to examine the pharmacokinetics of the absorption of radiolabeled AFB1 in rats in the presence of a yeast cell wall-based adsorbent (YCW) compared with that in the presence of the clay-based binder hydrated sodium calcium aluminosilicate (HSCAS). The results of the initial pharmacokinetic analysis showed that the absorption process across the GI tract was relatively slow, occurring over a matter of hours rather than minutes. The inclusion of mycotoxin binders increased the recovery of radiolabeled AFB1 in the small intestine, cecum, and colon at 5 and 10 h, revealing that they prevented AFB1 absorption compared with a control diet. Additionally, the accumulation of radiolabeled AFB1 was more significant in the blood plasma, kidney, and liver of animals fed the control diet, again showing the ability of the binders to reduce the assimilation of AFB1 into the body. The results showed the potential of YCW in reducing the absorption of AFB1 in vivo, and in protecting against the damaging effects of AFB1 contamination. Full article

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20 pages, 2506 KiB

Open AccessEditor’s ChoiceArticle

Efficient Aflatoxin B1 Sequestration by Yeast Cell Wall Extract and Hydrated Sodium Calcium Aluminosilicate Evaluated Using a Multimodal In-Vitro and Ex-Vivo Methodology

by Alexandros Yiannikouris, Juha Apajalahti, Hannele Kettunen, Suvi Ojanperä, Andrew N. W. Bell, Jason D. Keegan and Colm A. Moran

Toxins 2021, 13(1), 24; https://doi.org/10.3390/toxins13010024 - 01 Jan 2021

Cited by 10 | Viewed by 3077

Abstract

In this work, adsorption of the carcinogenic mycotoxin aflatoxin B1 (AFB1) by two sequestrants—a yeast cell wall-based adsorbent (YCW) and a hydrated sodium calcium aluminosilicate (HSCAS)—was studied across four laboratory models: (1) an in vitro model from a reference method was employed to [...] Read more.

In this work, adsorption of the carcinogenic mycotoxin aflatoxin B1 (AFB1) by two sequestrants—a yeast cell wall-based adsorbent (YCW) and a hydrated sodium calcium aluminosilicate (HSCAS)—was studied across four laboratory models: (1) an in vitro model from a reference method was employed to quantify the sorption capabilities of both sequestrants under buffer conditions at two pH values using liquid chromatography with fluorescence detection (LC-FLD); (2) in a second in vitro model, the influence of the upper gastrointestinal environment on the mycotoxin sorption capacity of the same two sequestrants was studied using a chronic AFB1 level commonly encountered in the field (10 µg/L and in the presence of feed); (3) the third model used a novel ex vivo approach to measure the absorption of ³H-labelled AFB1 in the intestinal tissue and the ability of the sequestrants to offset this process; and (4) a second previously developed ex vivo model readapted to AFB1 was used to measure the transfer of ³H-labelled AFB1 through live intestinal tissue, and the influence of sequestrants on its bioavailability by means of an Ussing chamber system. Despite some sorption effects caused by the feed itself studied in the second model, both in vitro models established that the adsorption capacity of both YCW and HSCAS is promoted at a low acidic pH. Ex vivo Models 3 and 4 showed that the same tested material formed a protective barrier on the epithelial mucosa and that they significantly reduced the transfer of AFB1 through live intestinal tissue. The results indicate that, by reducing the transmembrane transfer rate and reducing over 60% of the concentration of free AFB1, both products are able to significantly limit the bioavailability of AFB1. Moreover, there were limited differences between YCW and HSCAS in their sorption capacities. The inclusion of YCW in the dietary ration could have a positive influence in reducing AFB1′s physiological bioavailability. Full article

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2020

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27 pages, 427 KiB

Open AccessArticle

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

by Sara Franco Ortega, Ilenia Siciliano, Simona Prencipe, Maria Lodovica Gullino and Davide Spadaro

Toxins 2020, 12(12), 757; https://doi.org/10.3390/toxins12120757 - 30 Nov 2020

Cited by 10 | Viewed by 2839

Abstract

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that [...] Read more.

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan^® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts. Full article

17 pages, 1176 KiB

Open AccessArticle

Aflatoxin Reduction in Maize by Industrial-Scale Cleaning Solutions

by Michelangelo Pascale, Antonio F. Logrieco, Matthias Graeber, Marina Hirschberger, Mareike Reichel, Vincenzo Lippolis, Annalisa De Girolamo, Veronica M.T. Lattanzio and Katarina Slettengren

Toxins 2020, 12(5), 331; https://doi.org/10.3390/toxins12050331 - 17 May 2020

Cited by 19 | Viewed by 4427

Abstract

Different batches of biomass/feed quality maize contaminated by aflatoxins were processed at the industrial scale (a continuous process and separate discontinuous steps) to evaluate the effect of different cleaning solutions on toxin reduction. The investigated cleaning solutions included: (i) mechanical size separation of [...] Read more.

Different batches of biomass/feed quality maize contaminated by aflatoxins were processed at the industrial scale (a continuous process and separate discontinuous steps) to evaluate the effect of different cleaning solutions on toxin reduction. The investigated cleaning solutions included: (i) mechanical size separation of coarse, small and broken kernels, (ii) removal of dust/fine particles through an aspiration channel, (iii) separation of kernels based on gravity and (iv) optical sorting of spatial and spectral kernel defects. Depending on the sampled fraction, dynamic or static sampling was performed according to the Commission Regulation No. 401/2006 along the entire cleaning process lines. Aflatoxin analyses of the water–slurry aggregate samples were performed according to the AOAC Official Method No. 2005.008 based on high-performance liquid chromatography and immunoaffinity column cleanup of the extracts. A significant reduction in aflatoxin content in the cleaned products, ranging from 65% to 84% with respect to the uncleaned products, was observed when continuous cleaning lines were used. Additionally, an overall aflatoxin reduction from 55% to 94% was obtained by combining results from separate cleaning steps. High levels of aflatoxins (up to 490 µg/kg) were found in the rejected fractions, with the highest levels in dust and in the rejected fractions from the aspirator and optical sorting. This study shows that a cleaning line combining both mechanical and optical sorting technologies provides an efficient solution for reducing aflatoxin contamination in maize. Full article

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21 pages, 2325 KiB

Open AccessArticle

AFB1 Induced Transcriptional Regulation Related to Apoptosis and Lipid Metabolism in Liver of Chicken

by Xueqin Liu, Shailendra Kumar Mishra, Tao Wang, Zhongxian Xu, Xiaoling Zhao, Yan Wang, Huadong Yin, Xiaolan Fan, Bo Zeng, Mingyao Yang, Deying Yang, Qingyong Ni, Yan Li, Mingwang Zhang, Qing Zhu, Feng Chen and Diyan Li

Toxins 2020, 12(5), 290; https://doi.org/10.3390/toxins12050290 - 04 May 2020

Cited by 33 | Viewed by 3948

Abstract

Aflatoxin B1 (AFB1) leads to a major risk to poultry and its residues in meat products can also pose serious threat to human health. In this study, after feeding 165-day-old Roman laying hens for 35 days, the toxic effects of aflatoxin B1 at [...] Read more.

Aflatoxin B1 (AFB1) leads to a major risk to poultry and its residues in meat products can also pose serious threat to human health. In this study, after feeding 165-day-old Roman laying hens for 35 days, the toxic effects of aflatoxin B1 at different concentrations were evaluated. The purpose of this study was to explore the mechanism of liver toxicosis responses to AFB1. We found that highly toxic group exposure resulted in liver fat deposition, increased interstitial space, and hepatocyte apoptosis in laying hens. Furthermore, a total of 164 differentially expressed lnRNAs and 186 differentially expressed genes were found to be highly correlated (Pearson Correlation Coefficient > 0.80, p-value < 0.05) by sequencing the transcriptome of control (CB) and highly toxic group (TB3) chickens. We also identify 29 differentially expressed genes and 19 miRNAs that have targeted regulatory relationships. Based on the liver cell apoptosis and fatty liver syndrome that this research focused on, we found that the highly toxic AFB1 led to dysregulation of the expression of PPARG and BCL6. They are cis-regulated by TU10057 and TU45776, respectively. PPARG was the target gene of gga-miR-301a-3p, gga-miR-301b-3p, and BCL6 was the target gene of gga-miR-190a-3p. In summary, highly toxic AFB1 affects the expression levels of protein-coding genes and miRNAs in the liver of Roman layer hens, as well as the expression level of long non-coding RNA in the liver, which upregulates the expression of PPARG and downregulates the expression of Bcl-6. Our study provides information on possible genetic regulatory networks in AFB1-induced hepatic fat deposition and hepatocyte apoptosis. Full article

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12 pages, 3737 KiB

Open AccessArticle

A Sensitive, Point-of-Care Detection of Small Molecules Based on a Portable Barometer: Aflatoxins In Agricultural Products

by Weiqi Zhang, Wenqin Wu, Chong Cai, Xiaofeng Hu, Hui Li, Yizhen Bai, Zhaowei Zhang and Peiwu Li

Toxins 2020, 12(3), 158; https://doi.org/10.3390/toxins12030158 - 03 Mar 2020

Cited by 9 | Viewed by 2903

Abstract

Sensitive and point-of-care detection of small toxic molecules plays a key role in food safety. Aflatoxin, a typical small toxic molecule, can cause serious healthcare and economic issues, thereby promoting the development of sensitive and point-of-care detection. Although ELISA is one of the [...] Read more.

Sensitive and point-of-care detection of small toxic molecules plays a key role in food safety. Aflatoxin, a typical small toxic molecule, can cause serious healthcare and economic issues, thereby promoting the development of sensitive and point-of-care detection. Although ELISA is one of the official detection methods, it cannot fill the gap between sensitivity and point-of-care application because it requires a large-scale microplate reader. To employ portable readers in food safety, Pt-catalysis has attracted increasing attention due to its portability and reliability. In this study, we developed a sensitive point-of-care aflatoxin detection (POCAD) method via a portable handheld barometer. We synthesized and characterized Au@PtNPs and Au@PtNPs conjugated with a second antibody (Au@PtNPs-IgG). A competitive immunoassay was established based on the homemade monoclonal antibody against aflatoxins. Au@PtNPs-IgG was used to catalyze the production of O₂ from H₂O₂ in a sealed vessel. The pressure of O₂ was then recorded by a handheld barometer. The aflatoxin concentration was inversely proportional to the pressure recorded via the barometer reading. After optimization, a limit of detection of 0.03 ng/mL and a linear range from 0.09 to 16.0 ng/mL were achieved. Recovery was recorded as 83.1%–112.0% along with satisfactory results regarding inner- and inter-assay precision (relative standard deviation, RSD < 6.4%). Little cross-reaction was observed. Additionally, the POCAD was validated by high-performance liquid chromatography (HPLC) by using peanut and corn samples. The portable POCAD exhibits strong potential for applications in the on-site detection of small toxic molecules to ensure food safety. Full article

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2019

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19 pages, 2627 KiB

Open AccessArticle

Genetic Profiling of Aspergillus Isolates with Varying Aflatoxin Production Potential from Different Maize-Growing Regions of Kenya

by Richard Dooso Oloo, Sheila Okoth, Peter Wachira, Samuel Mutiga, Phillis Ochieng, Leah Kago, Fredrick Nganga, Jean-Baka Domelevo Entfellner and Sita Ghimire

Toxins 2019, 11(8), 467; https://doi.org/10.3390/toxins11080467 - 09 Aug 2019

Cited by 15 | Viewed by 5984

Abstract

Highly toxigenic strains of Aspergillus flavus have been reported to frequently contaminate maize, causing fatal aflatoxin poisoning in Kenya. To gain insights into the environmental and genetic factors that influence toxigenicity, fungi (n = 218) that were culturally identified as A. flavus [...] Read more.

Highly toxigenic strains of Aspergillus flavus have been reported to frequently contaminate maize, causing fatal aflatoxin poisoning in Kenya. To gain insights into the environmental and genetic factors that influence toxigenicity, fungi (n = 218) that were culturally identified as A. flavus were isolated from maize grains samples (n = 120) from three regions of Kenya. The fungi were further characterized to confirm their identities using a PCR-sequence analysis of the internal transcribed spacer (ITS) region of rDNA which also revealed all of them to be A. flavus. A subset of 72 isolates representing ITS sequence-based phylogeny cluster and the agroecological origin of maize samples was constituted for subsequent analysis. The analysis of partial calmodulin gene sequences showed that the subset consisted of A. flavus (87%) and Aspergillus minisclerotigenes (13%). No obvious association was detected between the presence of seven aflatoxin biosynthesis genes and fungal species or region. However, the presence of the aflD and aflS genes showed some association with aflatoxin production. The assessment of toxigenicity showed higher aflatoxin production potential in A. minisclerotigenes isolates. Given that A. minisclerotigenes were mainly observed in maize samples from Eastern Kenya, a known aflatoxin hotspot, we speculate that production of copious aflatoxin is an adaptative trait of this recently discovered species in the region. Full article

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14 pages, 730 KiB

Open AccessEditor’s ChoiceArticle

Aflatoxin in Chili Peppers in Nigeria: Extent of Contamination and Control Using Atoxigenic Aspergillus flavus Genotypes as Biocontrol Agents

by Chibundu N. Ezekiel, Alejandro Ortega-Beltran, Eniola O. Oyedeji, Joseph Atehnkeng, Philip Kössler, Folasade Tairu, Irmgard Hoeschle-Zeledon, Petr Karlovsky, Peter J. Cotty and Ranajit Bandyopadhyay

Toxins 2019, 11(7), 429; https://doi.org/10.3390/toxins11070429 - 22 Jul 2019

Cited by 34 | Viewed by 6218

Abstract

Across sub-Saharan Africa, chili peppers are fundamental ingredients of many traditional dishes. However, chili peppers may contain unsafe aflatoxin concentrations produced by Aspergillus section Flavi fungi. Aflatoxin levels were determined in chili peppers from three states in Nigeria. A total of 70 samples [...] Read more.

Across sub-Saharan Africa, chili peppers are fundamental ingredients of many traditional dishes. However, chili peppers may contain unsafe aflatoxin concentrations produced by Aspergillus section Flavi fungi. Aflatoxin levels were determined in chili peppers from three states in Nigeria. A total of 70 samples were collected from farmers’ stores and local markets. Over 25% of the samples contained unsafe aflatoxin concentrations. The chili peppers were associated with both aflatoxin producers and atoxigenic Aspergillus flavus genotypes. Efficacy of an atoxigenic biocontrol product, Aflasafe, registered in Nigeria for use on maize and groundnut, was tested for chili peppers grown in three states. Chili peppers treated with Aflasafe accumulated significantly less aflatoxins than nontreated chili peppers. The results suggest that Aflasafe is a valuable tool for the production of safe chili peppers. Use of Aflasafe in chili peppers could reduce human exposure to aflatoxins and increase chances to commercialize chili peppers in premium local and international markets. This is the first report of the efficacy of any atoxigenic biocontrol product for controlling aflatoxin in a spice crop. Full article

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16 pages, 2106 KiB

Open AccessArticle

Separation and Purification of Aflatoxins by Centrifugal Partition Chromatography

by Gábor Endre, Zsófia Hegedüs, Adiyadolgor Turbat, Biljana Škrbić, Csaba Vágvölgyi and András Szekeres

Toxins 2019, 11(6), 309; https://doi.org/10.3390/toxins11060309 - 30 May 2019

Cited by 10 | Viewed by 4066

Abstract

Aflatoxins are mycotoxins that are produced by several species of filamentous fungi. In the European Union, the concentration limits for this group of mycotoxins in food and feed products are very low (on the order of parts per billion). Thus, relatively high amounts [...] Read more.

Aflatoxins are mycotoxins that are produced by several species of filamentous fungi. In the European Union, the concentration limits for this group of mycotoxins in food and feed products are very low (on the order of parts per billion). Thus, relatively high amounts of these substances in their pure forms are required as reference standards. Chromatographic techniques based on solid stationary phases are generally used to purify these molecules; however, liquid–liquid chromatographic separations may be a promising alternative. Therefore, this study proposes a liquid–liquid chromatographic method for the separation of four aflatoxins and impurities. To optimise the method, numerous biphasic solvent systems (chloroform-, acetone- and acetic acid-based systems) were tested and evaluated in terms of their effectiveness at partitioning aflatoxins; the toluene/acetic acid/water (30:24:50, v/v/v/%) system was found to be the most efficient for application in centrifugal partition chromatographic instrument. Using liquid–liquid instrumental separation, the four aflatoxins, namely B₁ (400 mg), B₂ (34 mg), G₁ (817 mg) and G₂ (100 mg), were successfully isolated with 96.3%–98.2% purity from 4.5 L of Aspergillus parasiticus fermented material in a 250 mL centrifugal partition chromatography column. The identities and purities of the purified components were confirmed, and the performance parameters of each separation step and the whole procedure was determined. The developed method could be effectively used to purify aflatoxins for analytical applications. Full article

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13 pages, 2226 KiB

Open AccessArticle

Multiple CH/π Interactions Maintain the Binding of Aflatoxin B₁ in the Active Cavity of Human Cytochrome P450 1A2

by Jun Wu, Sisi Zhu, Yunbo Wu, Tianqing Jiang, Lingling Wang, Jun Jiang, Jikai Wen and Yiqun Deng

Toxins 2019, 11(3), 158; https://doi.org/10.3390/toxins11030158 - 12 Mar 2019

Cited by 9 | Viewed by 2979

Abstract

Human cytochrome P450 1A2 (CYP1A2) is one of the key CYPs that activate aflatoxin B₁ (AFB₁), a notorious mycotoxin, into carcinogenic exo-8,9-epoxides (AFBO) in the liver. Although the structure of CYP1A2 is available, the mechanism of CYP1A2-specific binding to AFB [...] Read more.

Human cytochrome P450 1A2 (CYP1A2) is one of the key CYPs that activate aflatoxin B₁ (AFB₁), a notorious mycotoxin, into carcinogenic exo-8,9-epoxides (AFBO) in the liver. Although the structure of CYP1A2 is available, the mechanism of CYP1A2-specific binding to AFB₁ has not been fully clarified. In this study, we used calculation biology to predict a model of CYP1A2 with AFB₁, where Thr-124, Phe-125, Phe-226, and Phe-260 possibly participate in the specific binding. Site-directed mutagenesis was performed to construct mutants T124A, F125A, F226A, and F260A. Escherichia coli-expressed recombinant proteins T124A, F226A, and F260A had active structures, while F125A did not. This was evidenced by Fe²⁺∙Carbon monoxide (CO)-reduced difference spectra and circular dichroism spectroscopy. Mutant F125A was expressed in HEK293T cells. Steady kinetic assays showed that T124A had enhanced activity towards AFB₁, while F125A, F226A, and F260A were significantly reduced in their ability to activate AFB₁, implying that hydrogen bonds between Thr-124 and AFB₁ were not important for substrate-specific binding, whereas Phe-125, Phe-226, and Phe-260 were essential for the process. The computation simulation and experimental results showed that the three key CH/π interactions between Phe-125, Phe-226, or Phe-260 and AFB₁ collectively maintained the stable binding of AFB₁ in the active cavity of CYP1A2. Full article

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12 pages, 1999 KiB

Open AccessEditor’s ChoiceArticle

Inhibition of Aflatoxin Production by Paraquat and External Superoxide Dismutase in Aspergillus flavus

by Tomohiro Furukawa and Shohei Sakuda

Toxins 2019, 11(2), 107; https://doi.org/10.3390/toxins11020107 - 12 Feb 2019

Cited by 17 | Viewed by 3382

Abstract

Aflatoxin contamination of crops is a worldwide problem, and elucidation of the regulatory mechanism of aflatoxin production, for example relative to the oxidative–antioxidative system, is needed. Studies have shown that oxidative stress induced by reactive oxygen species promotes aflatoxin production. However, superoxide has [...] Read more.

Aflatoxin contamination of crops is a worldwide problem, and elucidation of the regulatory mechanism of aflatoxin production, for example relative to the oxidative–antioxidative system, is needed. Studies have shown that oxidative stress induced by reactive oxygen species promotes aflatoxin production. However, superoxide has been suggested to have the opposite effect. Here, we investigated the effects of the superoxide generator, paraquat, and externally added superoxide dismutase (SOD) on aflatoxin production in Aspergillus flavus. Paraquat with an IC₅₀ value of 54.9 µM inhibited aflatoxin production without affecting fungal growth. It increased cytosolic and mitochondrial superoxide levels and downregulated the transcription of aflatoxin biosynthetic cluster genes, including aflR, a key regulatory protein. The addition of bovine Cu/ZnSOD to the culture medium suppressed the paraquat-induced increase in superoxide levels, but it did not fully restore paraquat-inhibited aflatoxin production because bovine Cu/ZnSOD with an IC₅₀ value of 17.9 µg/mL itself inhibited aflatoxin production. Externally added bovine Cu/ZnSOD increased the SOD activity in fungal cell extracts and upregulated the transcription of genes encoding Cu/ZnSOD and alcohol dehydrogenase. These results suggest that intracellular accumulation of superoxide impairs aflatoxin production by downregulating aflR expression, and that externally added Cu/ZnSOD also suppresses aflatoxin production by a mechanism other than canonical superoxide elimination activity. Full article

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22 pages, 2880 KiB

Open AccessEditor’s ChoiceArticle

Biological System Responses of Dairy Cows to Aflatoxin B1 Exposure Revealed with Metabolomic Changes in Multiple Biofluids

by Qian Wang, Yangdong Zhang, Nan Zheng, Liya Guo, Xiaoming Song, Shengguo Zhao and Jiaqi Wang

Toxins 2019, 11(2), 77; https://doi.org/10.3390/toxins11020077 - 01 Feb 2019

Cited by 40 | Viewed by 4253

Abstract

Research on mycotoxins now requires a systematic study of post-exposure organisms. In this study, the effects of aflatoxin B1 (AFB1) on biofluids biomarkers were examined with metabolomics and biochemical tests. The results showed that milk concentration of aflatoxin M1 changed with the addition [...] Read more.

Research on mycotoxins now requires a systematic study of post-exposure organisms. In this study, the effects of aflatoxin B1 (AFB1) on biofluids biomarkers were examined with metabolomics and biochemical tests. The results showed that milk concentration of aflatoxin M1 changed with the addition or removal of AFB1. AFB1 significantly affected serum concentrations of superoxide dismutase (SOD) and malon dialdehyde (MDA), SOD/MDA, and the total antioxidant capacity. Significant differences of volatile fatty acids and NH₃-N were detected in the rumen fluid. Eighteen rumen fluid metabolites, 11 plasma metabolites, and 9 milk metabolites were significantly affected by the AFB1. These metabolites are mainly involved in the pathway of amino acids metabolism. Our results suggest that not only is the study of macro-indicators (milk composition and production) important, but that more attention should be paid to micro-indicators (biomarkers) when assessing the risks posed by mycotoxins to dairy cows. Full article

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15 pages, 3785 KiB

Open AccessEditor’s ChoiceArticle

Lactobacillus bulgaricus or Lactobacillus rhamnosus Suppresses NF-κB Signaling Pathway and Protects against AFB₁-Induced Hepatitis: A Novel Potential Preventive Strategy for Aflatoxicosis?

by Yuanyuan Chen, Ruirui Li, Qiaocheng Chang, Zhihao Dong, Huanmin Yang and Chuang Xu

Toxins 2019, 11(1), 17; https://doi.org/10.3390/toxins11010017 - 04 Jan 2019

Cited by 33 | Viewed by 4225

Abstract

Aflatoxin B₁ (AFB₁), a mycotoxin found in food and feed, is immunotoxic to animals and poses significant threat to the food industry and animal production. The primary target of AFB₁ is the liver. To overcome aflatoxin toxicity, probiotic-mediated detoxification [...] Read more.

Aflatoxin B₁ (AFB₁), a mycotoxin found in food and feed, is immunotoxic to animals and poses significant threat to the food industry and animal production. The primary target of AFB₁ is the liver. To overcome aflatoxin toxicity, probiotic-mediated detoxification has been proposed. In the present study, to investigate the protective effects and molecular mechanisms of Lactobacillus bulgaricus or Lactobacillus rhamnosus against liver inflammatory responses to AFB₁, mice were administered with AFB₁ (300 μg/kg) and/or Lactobacillus intragastrically for 8 weeks. AML12 cells were cultured and treated with AFB₁, BAY 11-7082 (an NF-κB inhibitor), and different concentrations of L. bulgaricus or L. rhamnosus. The body weight, liver index, histopathological changes, biochemical indices, cytokines, cytotoxicity, and activation of the NF-κB signaling pathway were measured. AFB₁ exposure caused changes in liver histopathology and biochemical functions, altered inflammatory response, and activated the NF-κB pathway. Supplementation of L. bulgaricus or L. rhamnosus significantly prevented AFB₁-induced liver injury and alleviated histopathological changes and inflammatory response by decreasing NF-κB p65 expression. The results of in vitro experiments revealed that L. rhamnosus evidently protected against AFB₁-induced inflammatory response and decreased NF-κB p65 expression when compared with L. bulgaricus. These findings indicated that AFB₁ exposure can cause inflammatory response by inducing hepatic injury, and supplementation of L. bulgaricus or L. rhamnosus can produce significant protective effect against AFB₁-induced liver damage and inflammatory response by regulating the activation of the NF-κB signaling pathway. Full article

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2018

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16 pages, 1956 KiB

Open AccessArticle

Occurrence and Levels of Aflatoxins in Fish Feeds and Their Potential Effects on Fish in Nyeri, Kenya

by Evalyn Wanjiru Mwihia, Paul Gichohi Mbuthia, Gunnar Sundstøl Eriksen, James K. Gathumbi, Joyce G. Maina, Stephen Mutoloki, Robert Maina Waruiru, Isaac Rumpel Mulei and Jan Ludvig Lyche

Toxins 2018, 10(12), 543; https://doi.org/10.3390/toxins10120543 - 17 Dec 2018

Cited by 42 | Viewed by 5980

Abstract

Aflatoxins are fungal metabolites that contaminate foods and feeds, causing adverse health effects in humans and animals. This study determined the occurrence of aflatoxins in fish feeds and their potential effects on fish. Eighty-one fish feeds were sampled from 70 farms and 8 [...] Read more.

Aflatoxins are fungal metabolites that contaminate foods and feeds, causing adverse health effects in humans and animals. This study determined the occurrence of aflatoxins in fish feeds and their potential effects on fish. Eighty-one fish feeds were sampled from 70 farms and 8 feed manufacturing plants in Nyeri, Kenya for aflatoxin analysis using competitive enzyme-linked immunosorbent assay. Fish were sampled from 12 farms for gross and microscopic pathological examination. Eighty-four percent of feeds sampled tested positive for aflatoxins, ranging from 1.8 to 39.7 µg/kg with a mean of 7.0 ± 8.3 µg/kg and the median of 3.6 µg/kg. Fifteen feeds (18.5%) had aflatoxins above the maximum allowable level in Kenya of 10 µg/kg. Homemade and tilapia feeds had significantly higher aflatoxin levels than commercial and trout feeds. Feeds containing maize bran and fish meal had significantly higher aflatoxin levels than those without these ingredients. Five trout farms (41.7%) had fish with swollen abdomens, and enlarged livers with white or yellow nodules, which microscopically had large dark basophilic hepatic cells with hyperchromatic nuclei in irregular cords. In conclusion, aflatoxin contamination of fish feeds is prevalent in Nyeri, and may be the cause of adverse health effects in fish in this region. Full article

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11 pages, 1041 KiB

Open AccessArticle

Chestnut Drying Is Critical in Determining Aspergillus flavus Growth and Aflatoxin Contamination

by Simona Prencipe, Ilenia Siciliano, Carlotta Gatti, Maria Lodovica Gullino, Angelo Garibaldi and Davide Spadaro

Toxins 2018, 10(12), 530; https://doi.org/10.3390/toxins10120530 - 11 Dec 2018

Cited by 10 | Viewed by 3678

Abstract

Chestnut drying is used to prevent postharvest losses and microorganism contamination during storage. Several studies reported the contamination by aflatoxins (AFs) produced by Aspergillus spp. in chestnuts. The effect of drying temperatures (from 30 to 50 °C) was evaluated on the growth of [...] Read more.

Chestnut drying is used to prevent postharvest losses and microorganism contamination during storage. Several studies reported the contamination by aflatoxins (AFs) produced by Aspergillus spp. in chestnuts. The effect of drying temperatures (from 30 to 50 °C) was evaluated on the growth of A. flavus and the production of aflatoxins in chestnuts. The influence of the treatment on the proximate composition, the total phenol content and antioxidant activity of chestnuts was considered. Fungal colonization was observed on the nuts dried at 30, 35, and 40 °C; the incidence was lower at 40 °C. The highest concentrations of AFB₁ and AFB₂ were produced at 40 °C. No aflatoxins were detected at 45 or 50 °C. At 40 °C A. flavus was under suboptimal conditions for growth (a_w 0.78), but the fungus was able to synthesize aflatoxins. As the temperatures applied increased, the total phenol content increased, while the antioxidant activity decreased. A drying treatment at 45 °C for seven days (a_w 0.64) could be a promising method to effectively control both the growth of aflatoxigenic fungi and the production of aflatoxins. This study provides preliminary data useful to improve the current drying conditions used in chestnut mills, to reduce both fungal growth and aflatoxin production. Full article

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14 pages, 3770 KiB

Open AccessArticle

Aspergillus flavus NRRL 3251 Growth, Oxidative Status, and Aflatoxins Production Ability In Vitro under Different Illumination Regimes

by Tihomir Kovač, Bojan Šarkanj, Biljana Crevar, Marija Kovač, Ante Lončarić, Ivica Strelec, Chibundu N. Ezekiel, Michael Sulyok and Rudolf Krska

Toxins 2018, 10(12), 528; https://doi.org/10.3390/toxins10120528 - 10 Dec 2018

Cited by 14 | Viewed by 3584

Abstract

Aspergillus flavus is the most important mycotoxin-producing fungus involved in the global episodes of aflatoxin B₁ contamination of crops at both the pre-harvest and post-harvest stages. However, in order to effectively control aflatoxin contamination in crops using antiaflatoxigenic and/or antifungal compounds, some [...] Read more.

Aspergillus flavus is the most important mycotoxin-producing fungus involved in the global episodes of aflatoxin B₁ contamination of crops at both the pre-harvest and post-harvest stages. However, in order to effectively control aflatoxin contamination in crops using antiaflatoxigenic and/or antifungal compounds, some of which are photosensitive, a proper understanding of the photo-sensitive physiology of potential experimental strains need to be documented. The purpose of the study is therefore to evaluate the effect of visible (VIS) light illumination on growth and conidiation, aflatoxin production ability and modulation of A. flavus oxidative status during in vitro experiment. Aflatoxigenic A. flavus strain was inoculated in aflatoxin-inducing YES media and incubated under three different VIS illumination regimes during a 168 h growth period at 29 °C. VIS illumination reduced A. flavus mycelia biomass yield, both during growth on plates and in liquid media, promoted conidiation and increased the aflatoxin production. Furthermore, aflatoxin production increased with increased reactive oxidative species (ROS) levels at 96 h of growth, confirming illumination-driven oxidative stress modulation activity on A. flavus cells. Full article

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19 pages, 2752 KiB

Open AccessArticle

Alternative Splicing of the Aflatoxin-Associated Baeyer–Villiger Monooxygenase from Aspergillus flavus: Characterisation of MoxY Isoforms

by Carmien Tolmie, Martha S. Smit and Diederik J. Opperman

Toxins 2018, 10(12), 521; https://doi.org/10.3390/toxins10120521 - 05 Dec 2018

Cited by 3 | Viewed by 3312

Abstract

Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type [...] Read more.

Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type I Baeyer–Villiger monooxygenase (BVMO), responsible for the conversion of hydroxyversicolorone (HVN) and versicolorone (VN) to versiconal hemiacetal acetate (VHA) and versiconol acetate (VOAc), respectively. Using mRNA data, an intron near the C-terminus was identified that is alternatively spliced, creating two possible MoxY isoforms which exist in vivo, while analysis of the genomic DNA suggests an alternative start codon leading to possible elongation of the N-terminus. These four variants of the moxY gene were recombinantly expressed in Escherichia coli, and their activity evaluated with respect to their natural substrates HVN and VN, as well as surrogate ketone substrates. Activity of the enzyme is absolutely dependent on the additional 22 amino acid residues at the N-terminus. Two MoxY isoforms with alternative C-termini, MoxYAltN and MoxYAltNC, converted HVN and VN, in addition to a range of ketone substrates. Stability and flavin-binding data suggest that MoxYAltN is, most likely, the dominant isoform. MoxYAltNC is generated by intron splicing, in contrast to intron retention, which is the most prevalent type of alternative splicing in ascomycetes. The alternative C-termini did not alter the substrate acceptance profile, or regio- or enantioselectivity of the enzyme, but did significantly affect the solubility and stability. Full article

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15 pages, 1161 KiB

Open AccessArticle

Aspergillus texensis: A Novel Aflatoxin Producer with S Morphology from the United States

by Pummi Singh, Marc J. Orbach and Peter J. Cotty

Toxins 2018, 10(12), 513; https://doi.org/10.3390/toxins10120513 - 03 Dec 2018

Cited by 29 | Viewed by 4482

Abstract

Aflatoxins are carcinogenic metabolites produced primarily by fungi within Aspergillus section Flavi. These fungi infect a wide range of crops in warm regions. Molecular phylogenetic analyses of fungi with S morphology (average sclerotium size < 400 µm) within section Flavi collected from [...] Read more.

Aflatoxins are carcinogenic metabolites produced primarily by fungi within Aspergillus section Flavi. These fungi infect a wide range of crops in warm regions. Molecular phylogenetic analyses of fungi with S morphology (average sclerotium size < 400 µm) within section Flavi collected from across the United States (US) resulted in the discovery of a novel aflatoxin-producing species, Aspergillus texensis. Aspergillus texensis was isolated from maize grown in Arkansas, Louisiana, and Texas, and from soils cropped to maize in Texas. Aspergillus texensis produces sparse conidia and abundant sclerotia on various culture media, and on maize. Physiological studies have revealed optimal growth on culture media at 35 °C. All isolates of A. texensis produced B and G aflatoxins, cyclopiazonic acid and aspergillic acid. Aspergillus texensis and A. flavus S strain morphotypes produced similar concentrations of total aflatoxins on maize (p > 0.05). Phylogenetic analyses of aflatoxin-producers based on partial gene sequences of the β-tubulin (0.9 kb), calmodulin (1.2 kb), and nitrate reductase (2.1 kb) genes placed A. texensis in a highly supported monophyletic clade closely related to A. minisclerotigenes and a previously reported unnamed lineage designated Lethal Aflatoxicosis Fungus. Full article

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13 pages, 2362 KiB

Open AccessArticle

Streptomyces roseolus, A Promising Biocontrol Agent Against Aspergillus flavus, the Main Aflatoxin B₁ Producer

by Isaura Caceres, Selma P. Snini, Olivier Puel and Florence Mathieu

Toxins 2018, 10(11), 442; https://doi.org/10.3390/toxins10110442 - 30 Oct 2018

Cited by 27 | Viewed by 3866

Abstract

Crop contamination by aflatoxin B₁ is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute [...] Read more.

Crop contamination by aflatoxin B₁ is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute to the substitution of phytopharmaceutical products are thus needed. For this, a promising method resides in the use of biocontrol agents. Several actinobacteria strains have demonstrated to effectively reduce the aflatoxin B₁ concentration. Nevertheless, the molecular mechanism of action by which these biological agents reduce the mycotoxin concentration has not been determined. The aim of the present study was to test the potential use of Streptomyces roseolus as a biocontrol agent against aflatoxin B₁ contamination. Co-cultures with Aspergillus flavus were conducted, and the molecular fungal response was investigated through analyzing the q-PCR expression of 65 genes encoding relevant fungal functions. Moreover, kojic and cyclopiazonic acid concentrations, as well as morphological fungal changes were also analyzed. The results demonstrated that reduced concentrations of aflatoxin B₁ and kojic acid were respectively correlated with the down-regulation of the aflatoxin B₁ gene cluster and kojR gene expression. Moreover, a fungal hypersporulated phenotype and a general over-expression of genes involved in fungal development were observed in the co-culture condition. Full article

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15 pages, 1829 KiB

Open AccessArticle

Evaluation of Aflatoxin M1 Effects on the Metabolomic and Cytokinomic Profiling of a Hepatoblastoma Cell Line

by Silvia Marchese, Angela Sorice, Andrea Ariano, Salvatore Florio, Alfredo Budillon, Susan Costantini and Lorella Severino

Toxins 2018, 10(11), 436; https://doi.org/10.3390/toxins10110436 - 28 Oct 2018

Cited by 9 | Viewed by 2961

Abstract

Hepatoblastoma incidence has been associated with different environmental factors even if no data are reported about a correlation between aflatoxin exposure and hepatoblastoma initiation. Considering that hepatoblastoma develops in infants and children and aflatoxin M1 (AFM1), the aflatoxin B1 (AFB1) hydroxylated metabolite, can [...] Read more.

Hepatoblastoma incidence has been associated with different environmental factors even if no data are reported about a correlation between aflatoxin exposure and hepatoblastoma initiation. Considering that hepatoblastoma develops in infants and children and aflatoxin M1 (AFM1), the aflatoxin B1 (AFB1) hydroxylated metabolite, can be present in mothers’ milk and in marketed milk products, in this study we decided to test the effects of AFM1 on a hepatoblastoma cell line (HepG2). Firstly, we evaluated the effects of AFM1 on the cell viability, apoptosis, cell cycle, and metabolomic and cytokinomic profile of HepG2 cells after treatment. AFM1 induced: (1) a decrease of HepG2 cell viability, reaching IC₅₀ at 9 µM; (2) the blocking of the cell cycle in the G0/G1 phase; (3) the decrease of formiate levels and incremented level of some amino acids and metabolites in HepG2 cells after treatment; and (4) the increase of the concentration of three pro-inflammatory cytokines, IL-6, IL-8, and TNF-α, and the decrease of the anti-inflammatory interleukin, IL-4. Our results show that AFM1 inhibited the growth of HepG2 cells, inducing both a modulation of the lipidic, glycolytic, and amino acid metabolism and an increase of the inflammatory status of these cells. Full article

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16 pages, 4024 KiB

Open AccessArticle

The PHD Transcription Factor Rum1 Regulates Morphogenesis and Aflatoxin Biosynthesis in Aspergillus flavus

by Yule Hu, Guang Yang, Danping Zhang, Yaju Liu, Yu Li, Guanglan Lin, Zhiqiang Guo, Shihua Wang and Zhenhong Zhuang

Toxins 2018, 10(7), 301; https://doi.org/10.3390/toxins10070301 - 20 Jul 2018

Cited by 29 | Viewed by 3803

Abstract

Aspergillus flavus produces mycotoxins especially aflatoxin B₁ and infects crops worldwide. As a PHD transcription factor, there is no report on the role of Rum1 in the virulence of Aspergillus spp. yet. This study explored the biological function of Rum1 in A. [...] Read more.

Aspergillus flavus produces mycotoxins especially aflatoxin B₁ and infects crops worldwide. As a PHD transcription factor, there is no report on the role of Rum1 in the virulence of Aspergillus spp. yet. This study explored the biological function of Rum1 in A. flavus through the construction of rum1 deletion mutants and rum1 complementation strains with the method of homologous recombination. It was found, in the study, that Rum1 negatively regulates conidiation through abaA and brlA, positively regulates sclerotia formation through nsdC, nsdD, and sclR, triggers aflatoxin biological synthesis, and enhances the activity of amylase. Our findings suggested that Rum1 plays a major role in the growth of mycelia, conidia, and sclerotia production along with aflatoxin biosynthesis in A. flavus. Full article

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10 pages, 3363 KiB

Open AccessArticle

Detection of Aflatoxigenic and Atoxigenic Mexican Aspergillus Strains by the Dichlorvos–Ammonia (DV–AM) Method

by Masayo Kushiro, Hidemi Hatabayashi, Kimiko Yabe and Alexander Loladze

Toxins 2018, 10(7), 263; https://doi.org/10.3390/toxins10070263 - 27 Jun 2018

Cited by 8 | Viewed by 5199

Abstract

The dichlorvos–ammonia (DV–AM) method is a sensitive method for distinguishing aflatoxigenic fungi by detecting red (positive) colonies. In this study, the DV–AM method was applied for the isolation of aflatoxigenic and atoxigenic fungi from soil samples from a maize field in Mexico. In [...] Read more.

The dichlorvos–ammonia (DV–AM) method is a sensitive method for distinguishing aflatoxigenic fungi by detecting red (positive) colonies. In this study, the DV–AM method was applied for the isolation of aflatoxigenic and atoxigenic fungi from soil samples from a maize field in Mexico. In the first screening, we obtained two isolates from two soil subsamples of 20 independent samples and, in the second screening, we obtained two isolates from one subsample of these. Morphological and phylogenic analyses of the two isolates (MEX-A19-13, MEX-A19-2^nd-5) indicated that they were Aspergillus flavus located in the A. flavus clade. Chemical analyses demonstrated that one isolate could produce B-type aflatoxins, while the other produced no aflatoxins. These results demonstrate that the DV–AM method is useful for the isolation of both aflatoxigenic and atoxigenic Aspergilli. Full article

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16 pages, 2715 KiB

Open AccessArticle

Analysis of the Relationship between Alternative Respiration and Sterigmatocystin Formation in Aspergillus nidulans

by Ákos P. Molnár, Zoltán Németh, Erzsébet Fekete, Michel Flipphi, Nancy P. Keller and Levente Karaffa

Toxins 2018, 10(4), 168; https://doi.org/10.3390/toxins10040168 - 20 Apr 2018

Cited by 12 | Viewed by 5949

Abstract

Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of [...] Read more.

Aspergillus nidulans has one gene for alternative oxidase (EC 1.10.3.11). To investigate the relationship between this mitochondrial terminal oxidase and the formation of the mycotoxin sterigmatocystin, the encoding aodA gene was both deleted and overexpressed. Relative to the wild-type, the cyanide-resistant fraction of respiration in the late stationary stage—when sterigmatocystin production occurs—doubled in the overexpressing mutant carrying three aodA gene copies, but decreased to 10% in the deletant. Essentially identical results were obtained regardless whether the cultures were illuminated or protected from light. In contrast, sterigmatocystin yield in the aodA deletant was about half of that in the control when grown in the dark, while aodA overexpression resulted in up to 70% more sterigmatocystin formed, the yield increasing with alternative oxidase activity. Results were quite different when cultures were illuminated: under those conditions, sterigmatocystin volumetric yields were considerably lower, and statistically unvarying, regardless of the presence, absence, or the copy number of aodA. We conclude that the copy number of aodA, and hence, the balance between alternative- and cytochrome C-mediated respiration, appears to correlate with sterigmatocystin production in A. nidulans, albeit only in the absence of light. Full article

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15 pages, 4978 KiB

Open AccessArticle

G Protein α Subunit GpaB is Required for Asexual Development, Aflatoxin Biosynthesis and Pathogenicity by Regulating cAMP Signaling in Aspergillus flavus

by Yinghang Liu, Kunlong Yang, Qiuping Qin, Guinan Lin, Tianran Hu, Zhangling Xu and Shihua Wang

Toxins 2018, 10(3), 117; https://doi.org/10.3390/toxins10030117 - 10 Mar 2018

Cited by 22 | Viewed by 4666

Abstract

The heterotrimeric G proteins are critical for signal transduction and function in numerous biological processes including vegetative growth, asexual development and fungal virulence in fungi. Here, we identified four G protein alpha subunits (GanA, GpaB, FadA and GaoC) in the notorious Aflatoxin-producing fungus [...] Read more.

The heterotrimeric G proteins are critical for signal transduction and function in numerous biological processes including vegetative growth, asexual development and fungal virulence in fungi. Here, we identified four G protein alpha subunits (GanA, GpaB, FadA and GaoC) in the notorious Aflatoxin-producing fungus Aspergillus flavus. GanA, GpaB and FadA have homologues in other fungal species, while GaoC is a novel one. Here, we showed that the loss function of gpaB displayed a defect in conidiophore formation and considerably reduced expression levels of conidia-specific genes brlA and abaA. A decreased viability of cell wall integrity stress and oxidative stress were also found in the ∆gpaB mutant. More importantly, aflatoxin (AF) biosynthesis and infection on crop seeds were severely impaired in the gpaB-deficient mutant. Further analyses demonstrated that the intracellular cAMP levels significantly reduced in the gpaB-deficient mutant compared to wildtype strains. Additionally, an alteration of PKA activities in the ∆gpaB mutant was also found. Overall, our results indicated that GpaB played diverse roles in asexual sporulation, AF biosynthesis and virulence by regulating cAMP signaling in Aspergillus flavus. Full article

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10 pages, 746 KiB

Open AccessArticle

Survey of Candidate Genes for Maize Resistance to Infection by Aspergillus flavus and/or Aflatoxin Contamination

by Leigh K. Hawkins, Marilyn L. Warburton, Juliet D. Tang, John Tomashek, Dafne Alves Oliveira, Oluwaseun F. Ogunola, J. Spencer Smith and W. Paul Williams

Toxins 2018, 10(2), 61; https://doi.org/10.3390/toxins10020061 - 31 Jan 2018

Cited by 15 | Viewed by 5055

Abstract

Many projects have identified candidate genes for resistance to aflatoxin accumulation or Aspergillus flavus infection and growth in maize using genetic mapping, genomics, transcriptomics and/or proteomics studies. However, only a small percentage of these candidates have been validated in field conditions, and their [...] Read more.

Many projects have identified candidate genes for resistance to aflatoxin accumulation or Aspergillus flavus infection and growth in maize using genetic mapping, genomics, transcriptomics and/or proteomics studies. However, only a small percentage of these candidates have been validated in field conditions, and their relative contribution to resistance, if any, is unknown. This study presents a consolidated list of candidate genes identified in past studies or in-house studies, with descriptive data including genetic location, gene annotation, known protein identifiers, and associated pathway information, if known. A candidate gene pipeline to test the phenotypic effect of any maize DNA sequence on aflatoxin accumulation resistance was used in this study to determine any measurable effect on polymorphisms within or linked to the candidate gene sequences, and the results are published here. Full article

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24 pages, 1405 KiB

Open AccessArticle

Comparative Response of the Hepatic Transcriptomes of Domesticated and Wild Turkey to Aflatoxin B₁

by Kent M. Reed, Kristelle M. Mendoza, Juan E. Abrahante and Roger A. Coulombe

Toxins 2018, 10(1), 42; https://doi.org/10.3390/toxins10010042 - 13 Jan 2018

Cited by 14 | Viewed by 4588

Abstract

The food-borne mycotoxin aflatoxin B₁ (AFB₁) poses a significant risk to poultry, which are highly susceptible to its hepatotoxic effects. Domesticated turkeys (Meleagris gallopavo) are especially sensitive, whereas wild turkeys (M. g. silvestris) are more resistant. [...] Read more.

The food-borne mycotoxin aflatoxin B₁ (AFB₁) poses a significant risk to poultry, which are highly susceptible to its hepatotoxic effects. Domesticated turkeys (Meleagris gallopavo) are especially sensitive, whereas wild turkeys (M. g. silvestris) are more resistant. AFB₁ toxicity entails bioactivation by hepatic cytochrome P450s to the electrophilic exo-AFB₁-8,9-epoxide (AFBO). Domesticated turkeys lack functional hepatic GST-mediated detoxification of AFBO, and this is largely responsible for the differences in resistance between turkey types. This study was designed to characterize transcriptional changes induced in turkey livers by AFB₁, and to contrast the response of domesticated (susceptible) and wild (more resistant) birds. Gene expression responses to AFB₁ were examined using RNA-sequencing. Statistically significant differences in gene expression were observed among treatment groups and between turkey types. Expression analysis identified 4621 genes with significant differential expression (DE) in AFB₁-treated birds compared to controls. Characterization of DE transcripts revealed genes dis-regulated in response to toxic insult with significant association of Phase I and Phase II genes and others important in cellular regulation, modulation of apoptosis, and inflammatory responses. Constitutive expression of GSTA3 was significantly higher in wild birds and was significantly higher in AFB₁-treated birds when compared to controls for both genetic groups. This pattern was also observed by qRT-PCR in other wild and domesticated turkey strains. Results of this study emphasize the differential response of these genetically distinct birds, and identify genes and pathways that are differentially altered in aflatoxicosis. Full article

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12 pages, 5076 KiB

Open AccessArticle

Light-Irradiation Wavelength and Intensity Changes Influence Aflatoxin Synthesis in Fungi

by Tadahiro Suzuki

Toxins 2018, 10(1), 31; https://doi.org/10.3390/toxins10010031 - 05 Jan 2018

Cited by 14 | Viewed by 4814

Abstract

Fungi respond to light irradiation by forming conidia and occasionally synthesizing mycotoxins. Several light wavelengths, such as blue and red, affect the latter. However, the relationship between light irradiation and mycotoxin synthesis varies depending on the fungal species or strain. This study focused [...] Read more.

Fungi respond to light irradiation by forming conidia and occasionally synthesizing mycotoxins. Several light wavelengths, such as blue and red, affect the latter. However, the relationship between light irradiation and mycotoxin synthesis varies depending on the fungal species or strain. This study focused on aflatoxin (AF), which is a mycotoxin, and the types of light irradiation that increase AF synthesis. Light-irradiation tests using the visible region indicated that blue wavelengths in the lower 500 nm region promoted AF synthesis. In contrast, red wavelengths of 660 nm resulted in limited significant changes compared with dark conditions. Irradiation tests with different intensity levels indicated that a low light intensity increased AF synthesis. For one fungal strain, light irradiation decreased the AF synthesis under all wavelength conditions. However, the decrease was mitigated by 525 nm low intensity irradiation. Thus, blue-green low intensity irradiation may increase AF synthesis in fungi. Full article

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1848 KiB

Open AccessArticle

Carbon Dioxide Mediates the Response to Temperature and Water Activity Levels in Aspergillus flavus during Infection of Maize Kernels

by Matthew K. Gilbert, Angel Medina, Brian M. Mack, Matthew D. Lebar, Alicia Rodríguez, Deepak Bhatnagar, Naresh Magan, Gregory Obrian and Gary Payne

Toxins 2018, 10(1), 5; https://doi.org/10.3390/toxins10010005 - 22 Dec 2017

Cited by 34 | Viewed by 6238

Abstract

Aspergillus flavus is a saprophytic fungus that may colonize several important crops, including cotton, maize, peanuts and tree nuts. Concomitant with A. flavus colonization is its potential to secrete mycotoxins, of which the most prominent is aflatoxin. Temperature, water activity (a_w) [...] Read more.

Aspergillus flavus is a saprophytic fungus that may colonize several important crops, including cotton, maize, peanuts and tree nuts. Concomitant with A. flavus colonization is its potential to secrete mycotoxins, of which the most prominent is aflatoxin. Temperature, water activity (a_w) and carbon dioxide (CO₂) are three environmental factors shown to influence the fungus-plant interaction, which are predicted to undergo significant changes in the next century. In this study, we used RNA sequencing to better understand the transcriptomic response of the fungus to a_w, temperature, and elevated CO₂ levels. We demonstrate that aflatoxin (AFB₁) production on maize grain was altered by water availability, temperature and CO₂. RNA-Sequencing data indicated that several genes, and in particular those involved in the biosynthesis of secondary metabolites, exhibit different responses to water availability or temperature stress depending on the atmospheric CO₂ content. Other gene categories affected by CO₂ levels alone (350 ppm vs. 1000 ppm at 30 °C/0.99 a_w), included amino acid metabolism and folate biosynthesis. Finally, we identified two gene networks significantly influenced by changes in CO₂ levels that contain several genes related to cellular replication and transcription. These results demonstrate that changes in atmospheric CO₂ under climate change scenarios greatly influences the response of A. flavus to water and temperature when colonizing maize grain. Full article

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3858 KiB

Open AccessArticle

Aspergillus korhogoensis, a Novel Aflatoxin Producing Species from the Côte d’Ivoire

by Amaranta Carvajal-Campos, Ama Lethicia Manizan, Souria Tadrist, David Koffi Akaki, Rose Koffi-Nevry, Geromy G. Moore, Stephen O. Fapohunda, Sylviane Bailly, Didier Montet, Isabelle P. Oswald, Sophie Lorber, Catherine Brabet and Olivier Puel

Toxins 2017, 9(11), 353; https://doi.org/10.3390/toxins9110353 - 31 Oct 2017

Cited by 32 | Viewed by 8590

Abstract

Several strains of a new aflatoxigenic species of Aspergillus, A. korhogoensis, were isolated in the course of a screening study involving species from section Flavi found contaminating peanuts (Arachis hypogaea) and peanut paste in the Côte d’Ivoire. Based on [...] Read more.

Several strains of a new aflatoxigenic species of Aspergillus, A. korhogoensis, were isolated in the course of a screening study involving species from section Flavi found contaminating peanuts (Arachis hypogaea) and peanut paste in the Côte d’Ivoire. Based on examination of four isolates, this new species is described using a polyphasic approach. A concatenated alignment comprised of nine genes (ITS, benA, cmdA, mcm7, amdS, rpb1, preB, ppgA, and preA) was subjected to phylogenetic analysis, and resulted in all four strains being inferred as a distinct clade. Characterization of mating type for each strain revealed A. korhogoensis as a heterothallic species, since three isolates exhibited a singular MAT1-1 locus and one isolate exhibited a singular MAT1-2 locus. Morphological and physiological characterizations were also performed based on their growth on various types of media. Their respective extrolite profiles were characterized using LC/HRMS, and showed that this new species is capable of producing B- and G-aflatoxins, aspergillic acid, cyclopiazonic acid, aflavarins, and asparasones, as well as other metabolites. Altogether, our results confirm the monophyly of A. korhogoensis, and strengthen its position in the A. flavus clade, as the sister taxon of A. parvisclerotigenus. Full article

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3667 KiB

Open AccessArticle

Palladium Nanoparticles-Based Fluorescence Resonance Energy Transfer Aptasensor for Highly Sensitive Detection of Aflatoxin M₁ in Milk

by Hui Li, Daibin Yang, Peiwu Li, Qi Zhang, Wen Zhang, Xiaoxia Ding, Jin Mao and Jing Wu

Toxins 2017, 9(10), 318; https://doi.org/10.3390/toxins9100318 - 13 Oct 2017

Cited by 31 | Viewed by 6334

Abstract

A highly sensitive aptasensor for aflatoxin M₁ (AFM₁) detection was constructed based on fluorescence resonance energy transfer (FRET) between 5-carboxyfluorescein (FAM) and palladium nanoparticles (PdNPs). PdNPs (33 nm) were synthesized through a seed-mediated growth method and exhibited broad and strong [...] Read more.

A highly sensitive aptasensor for aflatoxin M₁ (AFM₁) detection was constructed based on fluorescence resonance energy transfer (FRET) between 5-carboxyfluorescein (FAM) and palladium nanoparticles (PdNPs). PdNPs (33 nm) were synthesized through a seed-mediated growth method and exhibited broad and strong absorption in the whole ultraviolet-visible (UV-Vis) range. The strong coordination interaction between nitrogen functional groups of the AFM₁ aptamer and PdNPs brought FAM and PdNPs in close proximity, which resulted in the fluorescence quenching of FAM to a maximum extent of 95%. The non-specific fluorescence quenching caused by PdNPs towards fluorescein was negligible. After the introduction of AFM₁ into the FAM-AFM₁ aptamer-PdNPs FRET system, the AFM₁ aptamer preferentially combined with AFM₁ accompanied by conformational change, which greatly weakened the coordination interaction between the AFM₁ aptamer and PdNPs. Thus, fluorescence recovery of FAM was observed and a linear relationship between the fluorescence recovery and the concentration of AFM₁ was obtained in the range of 5–150 pg/mL in aqueous buffer with the detection limit of 1.5 pg/mL. AFM₁ detection was also realized in milk samples with a linear detection range from 6 pg/mL to 150 pg/mL. The highly sensitive FRET aptasensor with simple configuration shows promising prospect in detecting a variety of food contaminants. Full article

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Open AccessArticle

The Putative Histone Methyltransferase DOT1 Regulates Aflatoxin and Pathogenicity Attributes in Aspergillus flavus

by Linlin Liang, Yinghang Liu, Kunlong Yang, Guinan Lin, Zhangling Xu, Huahui Lan, Xiuna Wang and Shihua Wang

Toxins 2017, 9(7), 232; https://doi.org/10.3390/toxins9070232 - 24 Jul 2017

Cited by 20 | Viewed by 5393

Abstract

Lysine methyltransferases transfer methyl groups in specific lysine sites, which regulates a variety of important biological processes in eukaryotes. In this study, we characterized a novel homolog of the yeast methyltransferase DOT1 in A. flavus, and observed the roles of dot1 [...] Read more.

Lysine methyltransferases transfer methyl groups in specific lysine sites, which regulates a variety of important biological processes in eukaryotes. In this study, we characterized a novel homolog of the yeast methyltransferase DOT1 in A. flavus, and observed the roles of dot1 in A. flavus. Deletion of dot1 showed a significant decrease in conidiation, but an increase in sclerotia formation. A change in viability to multiple stresses was also found in the Δdot1 mutant. Additionally, aflatoxin (AF) production was found severely impaired in the Δdot1 mutant. Further analysis by qRT-PCR revealed that the transcription of AF structural genes and their regulator gene aflS were prominently suppressed in the Δdot1 mutant. Furthermore, our data revealed that Dot1 is important for colonizing maize seeds in A. flavus. Our research indicates that Dot1 is involved in fungal development, aflatoxin biosynthesis and fungal virulence in A. flavus, which might provide a potential target for controlling A. flavus with new strategies. Full article

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Open AccessArticle

The Mode of Action of Cyclo(l-Ala-l-Pro) in Inhibiting Aflatoxin Production of Aspergillus flavus

by Kurin Iimura, Tomohiro Furukawa, Toshiyoshi Yamamoto, Lumi Negishi, Michio Suzuki and Shohei Sakuda

Toxins 2017, 9(7), 219; https://doi.org/10.3390/toxins9070219 - 12 Jul 2017

Cited by 16 | Viewed by 4676

Abstract

Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated [...] Read more.

Cyclo(l-Ala-l-Pro) inhibits aflatoxin production in aflatoxigenic fungi without affecting fungal growth. The mode of action of cyclo(l-Ala-l-Pro) in inhibiting aflatoxin production of Aspergillus flavus was investigated. A glutathione S-transferase (GST) of the fungus, designated AfGST, was identified as a binding protein of cyclo(l-Ala-l-Pro) in an experiment performed using cyclo(l-Ala-l-Pro)-immobilized Sepharose beads. Cyclo(l-Ala-l-Pro) specifically bound to recombinant AfGST and inhibited its GST activity. Ethacrynic acid, a known GST inhibitor, inhibited the GST activity of recombinant AfGST and aflatoxin production of the fungus. Ethacrynic acid reduced the expression level of AflR, a key regulatory protein for aflatoxin production, similar to cyclo(l-Ala-l-Pro). These results suggest that cyclo(l-Ala-l-Pro) inhibits aflatoxin production by affecting GST function in A. flavus, and that AfGST inhibitors are possible candidates as selective aflatoxin production inhibitors. Full article

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Open AccessArticle

Probing the Characterization of the Interaction of Aflatoxins B1 and G1 with Calf Thymus DNA In Vitro

by Liang Ma, Jiaman Wang and Yuhao Zhang

Toxins 2017, 9(7), 209; https://doi.org/10.3390/toxins9070209 - 01 Jul 2017

Cited by 25 | Viewed by 7622

Abstract

The binding characterization of aflatoxins with calf thymus DNA (ctDNA) under physiological conditions was investigated. Multispectroscopic techniques, ctDNA melting, viscosity measurements, and molecular docking techniques were employed to elucidate the binding mechanism of the aflatoxins with DNA. The fluorescence results indicated that both [...] Read more.

The binding characterization of aflatoxins with calf thymus DNA (ctDNA) under physiological conditions was investigated. Multispectroscopic techniques, ctDNA melting, viscosity measurements, and molecular docking techniques were employed to elucidate the binding mechanism of the aflatoxins with DNA. The fluorescence results indicated that both aflatoxin B1 (AFB1) and aflatoxin G1 (AFG1) bound to the ctDNA, forming complexes through hydrogen bonding. The binding constants of AFB1 and AFG1 with ctDNA reached up to 10³ L·mol⁻¹ and 10⁴ L·mol⁻¹, respectively, and AFG1 exhibited a higher binding propensity than that of AFB1. Furthermore, both AFB1 and AFG1 bound to the ctDNA through groove binding, as evidenced by the results of the spectroscopic, iodide quenching effect, viscosity, and ctDNA melting measurements. Changes in the circular dichroism signal manifested that both AFB1 and AFG1 induced an increase in the right-handed helicity, but only minimally influenced the base stacking of the DNA. A molecular docking study of the aflatoxin’s binding with the DNA revealed a groove binding mode, which was driven mainly by hydrogen bonding. This study of aflatoxin–ctDNA interaction may provide novel insights into the toxicological effect of the mycotoxins. Full article

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Open AccessArticle

Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells

by Yélian Marc Bossou, Youssra Serssar, Amel Allou, Sandrine Vitry, Isabelle Momas, Nathalie Seta, Jean Menotti and Sophie Achard

Toxins 2017, 9(7), 197; https://doi.org/10.3390/toxins9070197 - 22 Jun 2017

Cited by 19 | Viewed by 5129

Abstract

Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of [...] Read more.

Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of aflatoxin B₁ (AFB₁) and gliotoxin, two mycotoxins secreted by Aspergillus molds, on the biological activity of the human corneal epithelial (HCE) cells. After 24, 48, and 72 h of exposure, cellular viability and inflammatory response were assessed. Both endpoint cell viability colorimetric assays and continuous cell impedance measurements, providing noninvasive real-time assessment of the effect on cells, were performed. Cytokine gene expression and interleukin-8 release were quantified. Gliotoxin appeared more cytotoxic than AFB₁ but, at the same time, led to a lower increase of the inflammatory response reflecting its immunosuppressive properties. Real-time cell impedance measurement showed a distinct profile of cytotoxicity for both mycotoxins. HCE cells appeared to be a well-suited in vitro model to study ocular surface reactivity following biological contaminant exposure. Low, but persistent inflammation, caused by environmental factors, such as fungal toxins, leads to irritation and sensitization, and could be responsible for allergic manifestations which, in turn, could lead to mucosal hyper-reactivity. Full article

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Open AccessArticle

Aflatoxin B1 Tolerance and Accumulation in Black Soldier Fly Larvae (Hermetia illucens) and Yellow Mealworms (Tenebrio molitor)

by Guido Bosch, H. J. van der Fels-Klerx, Theo C. de Rijk and Dennis G. A. B. Oonincx

Toxins 2017, 9(6), 185; https://doi.org/10.3390/toxins9060185 - 02 Jun 2017

Cited by 97 | Viewed by 8829

Abstract

Crops contaminated with fungal mycotoxins such as aflatoxin B1 (AFB1) are often downgraded or removed from the food chain. This study aimed to evaluate the tolerance and accumulation of AFB1 in two insect species to determine whether they could be used to retain [...] Read more.

Crops contaminated with fungal mycotoxins such as aflatoxin B1 (AFB1) are often downgraded or removed from the food chain. This study aimed to evaluate the tolerance and accumulation of AFB1 in two insect species to determine whether they could be used to retain condemned mycotoxin contaminated crops in the food chain. First, instar black soldier fly larvae (Hermetia illucens, BSF) and yellow mealworm (Tenebrio molitor, YMW) were fed poultry feed spiked with AFB1 and formulated to contain levels of 0.01, 0.025, 0.05, 0.10, 0.25, and up to 0.5 mg/kg dry feed. Poultry feed without any additions and feed with only the solvent added served as controls. The AFB1 in the feed did not affect survival and body weight in the BSF and YMW larvae (p > 0.10), indicating a high tolerance to aflatoxin B1 in both species. Furthermore, AFB1 and aflatoxin M1 (AFM1) were below the detection limit (0.10 µg/kg) in BSF larvae, whereas the YMW had AFB1 levels that were approximately 10% of the European Union’s legal limit for feed materials and excreted AFM1. It is concluded that both BSF larvae and YMW have a high AFB1 tolerance and do not accumulate AFB1. Full article

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Open AccessArticle

Inhibitory Activities of Blasticidin S Derivatives on Aflatoxin Production by Aspergillus Flavus

by Tomoya Yoshinari, Yoshiko Sugita-Konishi, Takahiro Ohnishi and Jun Terajima

Toxins 2017, 9(6), 176; https://doi.org/10.3390/toxins9060176 - 26 May 2017

Cited by 7 | Viewed by 3818

Abstract

Blasticidin S (BcS) is a protein synthesis inhibitor which shows strong growth inhibitory activity against a number of microorganisms. However, BcS inhibited aflatoxin production by Aspergillus flavus without affecting its growth. In order to obtain information about the structure–activity relationship of BcS as [...] Read more.

Blasticidin S (BcS) is a protein synthesis inhibitor which shows strong growth inhibitory activity against a number of microorganisms. However, BcS inhibited aflatoxin production by Aspergillus flavus without affecting its growth. In order to obtain information about the structure–activity relationship of BcS as an aflatoxin production inhibitor, BcS derivatives were prepared and their aflatoxin production inhibitory activities were evaluated. Among five derivatives, blasticidin S carboxymethyl ester, deaminohydroxyblasticidin S, and pyrimidinoblasticidin S showed inhibitory activity, while the others did not. The IC₅₀ value for aflatoxin production of the carboxymethyl ester derivative was one-fifth of that of BcS although their antimicrobial activities were almost the same. These results indicate that the inhibitory activity of BcS against aflatoxin production was enhanced by esterification of its carboxyl group and that the carboxymethyl ester derivative might be more suitable for practical use than BcS because of the specificity of the carboxymethyl ester derivative, which inhibited aflatoxin production more than BcS. Full article

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Open AccessArticle

Doses of Immunogen Contribute to Specificity Spectrums of Antibodies against Aflatoxin

by Peiwu Li, Jing Wu, Li Zhang, Zhiyong Fan, Tingting Yu, Feng Jiang, Xiaoqian Tang, Zhaowei Zhang, Wen Zhang and Qi Zhang

Toxins 2017, 9(5), 172; https://doi.org/10.3390/toxins9050172 - 19 May 2017

Cited by 9 | Viewed by 4527

Abstract

Research about antibody specificity spectra was conducted to develop single-specific antibodies or broad-specific antibodies. Aflatoxins, as one class of high-toxicity mycotoxins, were selected as the research targets to investigate the effect of the immunogen dose on antibody specificity spectra. For this aim, 16 [...] Read more.

Research about antibody specificity spectra was conducted to develop single-specific antibodies or broad-specific antibodies. Aflatoxins, as one class of high-toxicity mycotoxins, were selected as the research targets to investigate the effect of the immunogen dose on antibody specificity spectra. For this aim, 16 monoclonal antibodies were induced by low or high doses of aflatoxin B₁-BSA, and 34 monoclonal antibodies were induced by low or high doses of aflatoxin M₁-BSA. The specificities of the antibodies induced, whether by aflatoxin B₁ conjugate or aflatoxin M₁ conjugate, indicated that the low dose of the immunogen induced a narrow spectrum of antibody specificity, while the high dose of the immunogen showed an advantage to form a broad spectrum of antibody specificity. Therefore, this report provides important information for the development of new antibodies against small molecules like aflatoxins. Full article

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Open AccessArticle

Aflatoxin B₁-Induced Developmental and DNA Damage in Caenorhabditis elegans

by Wei-Hong Feng, Kathy S. Xue, Lili Tang, Phillip L. Williams and Jia-Sheng Wang

Toxins 2017, 9(1), 9; https://doi.org/10.3390/toxins9010009 - 26 Dec 2016

Cited by 25 | Viewed by 5745

Abstract

Aflatoxin B₁ (AFB₁) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB₁ has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB₁ is also a [...] Read more.

Aflatoxin B₁ (AFB₁) is a ubiquitous mycotoxin produced by toxicogenic Aspergillus species. AFB₁ has been reported to cause serious adverse health effects, such as cancers and abnormal development and reproduction, in animals and humans. AFB₁ is also a potent genotoxic mutagen that causes DNA damage in vitro and in vivo. However, the link between DNA damage and abnormal development and reproduction is unclear. To address this issue, we examined the DNA damage, germline apoptosis, growth, and reproductive toxicity following exposure to AFB₁, using Caenorhabditis elegans as a study model. Results found that AFB₁ induced DNA damage and germline apoptosis, and significantly inhibited growth and reproduction of the nematodes in a concentration-dependent manner. Exposure to AFB₁ inhibited growth or reproduction more potently in the DNA repair-deficient xpa-1 nematodes than the wild-type N2 strain. According to the relative expression level of pathway-related genes measured by real-time PCR, the DNA damage response (DDR) pathway was found to be associated with AFB₁-induced germline apoptosis, which further played an essential role in the dysfunction of growth and reproduction in C. elegans. Full article

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Open AccessArticle

Growth-Phase Sterigmatocystin Formation on Lactose Is Mediated via Low Specific Growth Rates in Aspergillus nidulans

by Zoltán Németh, Ákos P. Molnár, Balázs Fejes, Levente Novák, Levente Karaffa, Nancy P. Keller and Erzsébet Fekete

Toxins 2016, 8(12), 354; https://doi.org/10.3390/toxins8120354 - 28 Nov 2016

Cited by 15 | Viewed by 4647

Abstract

Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, [...] Read more.

Seed contamination with polyketide mycotoxins such as sterigmatocystin (ST) produced by Aspergilli is a worldwide issue. The ST biosynthetic pathway is well-characterized in A. nidulans, but regulatory aspects related to the carbon source are still enigmatic. This is particularly true for lactose, inasmuch as some ST production mutant strains still synthesize ST on lactose but not on other carbon substrates. Here, kinetic data revealed that on d-glucose, ST forms only after the sugar is depleted from the medium, while on lactose, ST appears when most of the carbon source is still available. Biomass-specified ST production on lactose was significantly higher than on d-glucose, suggesting that ST formation may either be mediated by a carbon catabolite regulatory mechanism, or induced by low specific growth rates attainable on lactose. These hypotheses were tested by d-glucose limited chemostat-type continuous fermentations. No ST formed at a high growth rate, while a low growth rate led to the formation of 0.4 mg·L⁻¹ ST. Similar results were obtained with a CreA mutant strain. We concluded that low specific growth rates may be the primary cause of mid-growth ST formation on lactose in A. nidulans, and that carbon utilization rates likely play a general regulatory role during biosynthesis. Full article

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Open AccessArticle

RNA Sequencing of Contaminated Seeds Reveals the State of the Seed Permissive for Pre-Harvest Aflatoxin Contamination and Points to a Potential Susceptibility Factor

by Josh Clevenger, Kathleen Marasigan, Vasileios Liakos, Victor Sobolev, George Vellidis, Corley Holbrook and Peggy Ozias-Akins

Toxins 2016, 8(11), 317; https://doi.org/10.3390/toxins8110317 - 03 Nov 2016

Cited by 13 | Viewed by 5591

Abstract

Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for [...] Read more.

Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for PAC resistance has been slow. It has been shown that aflatoxin production can be induced by applying drought stress as peanut seeds mature. We have implemented an automated rainout shelter that controls temperature and moisture in the root and peg zone to induce aflatoxin production. Using polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC), seeds meeting the following conditions were selected: infected with Aspergillus flavus and contaminated with aflatoxin; and not contaminated with aflatoxin. RNA sequencing analysis revealed groups of genes that describe the transcriptional state of contaminated vs. uncontaminated seed. These data suggest that fatty acid biosynthesis and abscisic acid (ABA) signaling are altered in contaminated seeds and point to a potential susceptibility factor, ABR1, as a repressor of ABA signaling that may play a role in permitting PAC. Full article

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Open AccessArticle

Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B₁

by Jun Wu, Ruohong Chen, Caihui Zhang, Kangbai Li, Weiying Xu, Lijuan Wang, Qingmei Chen, Peiqiang Mu, Jun Jiang, Jikai Wen and Yiqun Deng

Toxins 2016, 8(9), 267; https://doi.org/10.3390/toxins8090267 - 12 Sep 2016

Cited by 20 | Viewed by 5291

Abstract

Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B₁ (AFB₁) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB₁ in pigs remains to be clarified, especially [...] Read more.

Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B₁ (AFB₁) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB₁ in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB₁ to form AFB₁-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB₁ by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB₁. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB₁ binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB₁. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB₁ in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB₁. Full article

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Open AccessArticle

Confirmation and Fine Mapping of a Major QTL for Aflatoxin Resistance in Maize Using a Combination of Linkage and Association Mapping

by Yu Zhang, Min Cui, Jimin Zhang, Lei Zhang, Chenliu Li, Xin Kan, Qian Sun, Dexiang Deng and Zhitong Yin

Toxins 2016, 8(9), 258; https://doi.org/10.3390/toxins8090258 - 02 Sep 2016

Cited by 20 | Viewed by 6266

Abstract

Maize grain contamination with aflatoxin from Aspergillus flavus (A. flavus) is a serious health hazard to animals and humans. To map the quantitative trait loci (QTLs) associated with resistance to A. flavus, we employed a powerful approach that differs from [...] Read more.

Maize grain contamination with aflatoxin from Aspergillus flavus (A. flavus) is a serious health hazard to animals and humans. To map the quantitative trait loci (QTLs) associated with resistance to A. flavus, we employed a powerful approach that differs from previous methods in one important way: it combines the advantages of the genome-wide association analysis (GWAS) and traditional linkage mapping analysis. Linkage mapping was performed using 228 recombinant inbred lines (RILs), and a highly significant QTL that affected aflatoxin accumulation, qAA8, was mapped. This QTL spanned approximately 7 centi-Morgan (cM) on chromosome 8. The confidence interval was too large for positional cloning of the causal gene. To refine this QTL, GWAS was performed with 558,629 single nucleotide polymorphisms (SNPs) in an association population comprising 437 maize inbred lines. Twenty-five significantly associated SNPs were identified, most of which co-localised with qAA8 and explained 6.7% to 26.8% of the phenotypic variation observed. Based on the rapid linkage disequilibrium (LD) and the high density of SNPs in the association population, qAA8 was further localised to a smaller genomic region of approximately 1500 bp. A high-resolution map of the qAA8 region will be useful towards a marker-assisted selection (MAS) of A. flavus resistance and a characterisation of the causal gene. Full article

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Open AccessArticle

The Stress Response Regulator AflSkn7 Influences Morphological Development, Stress Response, and Pathogenicity in the Fungus Aspergillus flavus

by Feng Zhang, Gaopo Xu, Longpo Geng, Xiaoyan Lu, Kunlong Yang, Jun Yuan, Xinyi Nie, Zhenhong Zhuang and Shihua Wang

Toxins 2016, 8(7), 202; https://doi.org/10.3390/toxins8070202 - 05 Jul 2016

Cited by 37 | Viewed by 6471

Abstract

This study focused on AflSkn7, which is a stress response regulator in the aflatoxin-producing Aspergillus flavus. The ΔAflSkn7 mutants exhibited partially defective conidial formation and a complete inability to generate sclerotia, indicating AflSkn7 affects A. flavus asexual and sexual development. The [...] Read more.

This study focused on AflSkn7, which is a stress response regulator in the aflatoxin-producing Aspergillus flavus. The ΔAflSkn7 mutants exhibited partially defective conidial formation and a complete inability to generate sclerotia, indicating AflSkn7 affects A. flavus asexual and sexual development. The mutants tolerated osmotic stress but were partially susceptible to the effects of cell wall stress. Additionally, the ΔAflSkn7 mutants were especially sensitive to oxidative stress. These observations confirmed that AflSkn7 influences oxidative stress responses rather than osmotic stress responses. Additionally, AflSkn7 was observed to increase aflatoxin biosynthesis and seed infection rates. These results indicate AflSkn7 affects A. flavus morphological development, stress response, aflatoxin production, and pathogenicity. The results of this study may facilitate the development of new methods to manage A. flavus infections. Full article

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Open AccessArticle

Inhibitory Activities of Alkyl Syringates and Related Compounds on Aflatoxin Production

by Tomohiro Furukawa, Kurin Iimura, Taichi Kimura, Toshiyoshi Yamamoto and Shohei Sakuda

Toxins 2016, 8(6), 177; https://doi.org/10.3390/toxins8060177 - 07 Jun 2016

Cited by 8 | Viewed by 4760

Abstract

Inhibitors of aflatoxin production of aflatoxigenic fungi are useful for preventing aflatoxin contamination in crops. As methyl syringate weakly inhibits aflatoxin production, aflatoxin production inhibitory activities of additional alkyl syringates with alkyl chains from ethyl to octyl were examined. Inhibitory activity toward aflatoxin [...] Read more.

Inhibitors of aflatoxin production of aflatoxigenic fungi are useful for preventing aflatoxin contamination in crops. As methyl syringate weakly inhibits aflatoxin production, aflatoxin production inhibitory activities of additional alkyl syringates with alkyl chains from ethyl to octyl were examined. Inhibitory activity toward aflatoxin production of Aspergillus flavus became stronger as the length of the alkyl chains on the esters became longer. Pentyl, hexyl, heptyl, and octyl syringates showed strong activity at 0.05 mM. Heptyl and octyl parabens, and octyl gallate also inhibited aflatoxin production as strongly as octyl syringate. Alkyl parabens and alkyl gallates inhibit the complex II activity of the mitochondrial respiration chain; thus, whether alkyl syringates inhibit complex II activity was examined. Inhibitory activities of alkyl syringates toward complex II also became stronger as the length of the alkyl chains increased. The complex II inhibitory activity of octyl syringate was comparable to that of octyl paraben and octyl gallate. These results suggest that alkyl syringates, alkyl parabens, and alkyl gallates, including commonly used food additives, are useful for aflatoxin control. Full article

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Open AccessArticle

Effects of Zinc Chelators on Aflatoxin Production in Aspergillus parasiticus

by Josephine Wee, Devin M. Day and John E. Linz

Toxins 2016, 8(6), 171; https://doi.org/10.3390/toxins8060171 - 02 Jun 2016

Cited by 9 | Viewed by 5986

Abstract

Zinc concentrations strongly influence aflatoxin accumulation in laboratory media and in food and feed crops. The presence of zinc stimulates aflatoxin production, and the absence of zinc impedes toxin production. Initial studies that suggested a link between zinc and aflatoxin biosynthesis were presented [...] Read more.

Zinc concentrations strongly influence aflatoxin accumulation in laboratory media and in food and feed crops. The presence of zinc stimulates aflatoxin production, and the absence of zinc impedes toxin production. Initial studies that suggested a link between zinc and aflatoxin biosynthesis were presented in the 1970s. In the present study, we utilized two zinc chelators, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and 2,3-dimercapto-1-propanesulfonic acid (DMPS) to explore the effect of zinc limitation on aflatoxin synthesis in Aspergillus parasiticus. TPEN but not DMPS decreased aflatoxin biosynthesis up to six-fold depending on whether A. parasiticus was grown on rich or minimal medium. Although we observed significant inhibition of aflatoxin production by TPEN, no detectable changes were observed in expression levels of the aflatoxin pathway gene ver-1 and the zinc binuclear cluster transcription factor, AflR. Treatment of growing A. parasiticus solid culture with a fluorescent zinc probe demonstrated an increase in intracellular zinc levels assessed by increases in fluorescent intensity of cultures treated with TPEN compared to controls. These data suggest that TPEN binds to cytoplasmic zinc therefore limiting fungal access to zinc. To investigate the efficacy of TPEN on food and feed crops, we found that TPEN effectively decreases aflatoxin accumulation on peanut medium but not in a sunflower seeds-derived medium. From an application perspective, these data provide the basis for biological differences that exist in the efficacy of different zinc chelators in various food and feed crops frequently contaminated by aflatoxin. Full article

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Open AccessArticle

Use of Cold Atmospheric Plasma to Detoxify Hazelnuts from Aflatoxins

by Ilenia Siciliano, Davide Spadaro, Ambra Prelle, Dario Vallauri, Maria Chiara Cavallero, Angelo Garibaldi and Maria Lodovica Gullino

Toxins 2016, 8(5), 125; https://doi.org/10.3390/toxins8050125 - 26 Apr 2016

Cited by 111 | Viewed by 8085

Abstract

Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to [...] Read more.

Aflatoxins, produced by Aspergillus flavus and A. parasiticus, can contaminate different foodstuffs, such as nuts. Cold atmospheric pressure plasma has the potential to be used for mycotoxin detoxification. In this study, the operating parameters of cold atmospheric pressure plasma were optimized to reduce the presence of aflatoxins on dehulled hazelnuts. First, the effect of different gases was tested (N₂, 0.1% O₂ and 1% O₂, 21% O₂), then power (400, 700, 1000, 1150 W) and exposure time (1, 2, 4, and 12 min) were optimized. In preliminary tests on aflatoxin standard solutions, this method allowed to obtain a complete detoxification using a high power for a few minutes. On hazelnuts, in similar conditions (1000 W, 12 min), a reduction in the concentration of total aflatoxins and AFB₁ of over 70% was obtained. Aflatoxins B₁ and G₁ were more sensitive to plasma treatments compared to aflatoxins B₂ and G₂, respectively. Under plasma treatment, aflatoxin B₁ was more sensitive compared to aflatoxin G₁. At the highest power, and for the longest time, the maximum temperature increment was 28.9 °C. Cold atmospheric plasma has the potential to be a promising method for aflatoxin detoxification on food, because it is effective and it could help to maintain the organoleptic characteristics. Full article

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Open AccessFeature PaperArticle

Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach

by Isaura Caceres, Rhoda El Khoury, Ángel Medina, Yannick Lippi, Claire Naylies, Ali Atoui, André El Khoury, Isabelle P. Oswald, Jean-Denis Bailly and Olivier Puel

Toxins 2016, 8(5), 123; https://doi.org/10.3390/toxins8050123 - 26 Apr 2016

Cited by 49 | Viewed by 6872

Abstract

Produced by several species of Aspergillus, Aflatoxin B₁ (AFB₁) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, [...] Read more.

Produced by several species of Aspergillus, Aflatoxin B₁ (AFB₁) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB₁ is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B₁ cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB₁ inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB₁ biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB₁ cluster regulation. Full article

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Open AccessArticle

Functional Genomic Analysis of Aspergillus flavus Interacting with Resistant and Susceptible Peanut

by Houmiao Wang, Yong Lei, Liying Yan, Liyun Wan, Xiaoping Ren, Silong Chen, Xiaofeng Dai, Wei Guo, Huifang Jiang and Boshou Liao

Toxins 2016, 8(2), 46; https://doi.org/10.3390/toxins8020046 - 15 Feb 2016

Cited by 25 | Viewed by 8480

Abstract

In the Aspergillus flavus (A. flavus)–peanut pathosystem, development and metabolism of the fungus directly influence aflatoxin contamination. To comprehensively understand the molecular mechanism of A. flavus interaction with peanut, RNA-seq was used for global transcriptome profiling of A. flavus during interaction with [...] Read more.

In the Aspergillus flavus (A. flavus)–peanut pathosystem, development and metabolism of the fungus directly influence aflatoxin contamination. To comprehensively understand the molecular mechanism of A. flavus interaction with peanut, RNA-seq was used for global transcriptome profiling of A. flavus during interaction with resistant and susceptible peanut genotypes. In total, 67.46 Gb of high-quality bases were generated for A. flavus-resistant (af_R) and -susceptible peanut (af_S) at one (T1), three (T2) and seven (T3) days post-inoculation. The uniquely mapped reads to A. flavus reference genome in the libraries of af_R and af_S at T2 and T3 were subjected to further analysis, with more than 72% of all obtained genes expressed in the eight libraries. Comparison of expression levels both af_R vs. af_S and T2 vs. T3 uncovered 1926 differentially expressed genes (DEGs). DEGs associated with mycelial growth, conidial development and aflatoxin biosynthesis were up-regulated in af_S compared with af_R, implying that A. flavus mycelia more easily penetrate and produce much more aflatoxin in susceptible than in resistant peanut. Our results serve as a foundation for understanding the molecular mechanisms of aflatoxin production differences between A. flavus-R and -S peanut, and offer new clues to manage aflatoxin contamination in crops. Full article

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Open AccessFeature PaperArticle

The Master Transcription Factor mtfA Governs Aflatoxin Production, Morphological Development and Pathogenicity in the Fungus Aspergillus flavus

by Zhenhong Zhuang, Jessica M. Lohmar, Timothy Satterlee, Jeffrey W. Cary and Ana M. Calvo

Toxins 2016, 8(1), 29; https://doi.org/10.3390/toxins8010029 - 20 Jan 2016

Cited by 55 | Viewed by 6692

Abstract

Aspergillus flavus produces a variety of toxic secondary metabolites; among them, the aflatoxins (AFs) are the most well known. These compounds are highly mutagenic and carcinogenic, particularly AFB₁. A. flavus is capable of colonizing a number of economically-important crops, such as [...] Read more.

Aspergillus flavus produces a variety of toxic secondary metabolites; among them, the aflatoxins (AFs) are the most well known. These compounds are highly mutagenic and carcinogenic, particularly AFB₁. A. flavus is capable of colonizing a number of economically-important crops, such as corn, cotton, peanut and tree nuts, and contaminating them with AFs. Molecular genetic studies in A. flavus could identify novel gene targets for use in strategies to reduce AF contamination and its adverse impact on food and feed supplies worldwide. In the current study, we investigated the role of the master transcription factor gene mtfA in A. flavus. Our results revealed that forced overexpression of mtfA results in a drastic decrease or elimination of several secondary metabolites, among them AFB₁. The reduction in AFB₁ was accompanied by a decrease in aflR expression. Furthermore, mtfA also regulates development; conidiation was influenced differently by this gene depending on the type of colonized substrate. In addition to its effect on conidiation, mtfA is necessary for the normal maturation of sclerotia. Importantly, mtfA positively affects the pathogenicity of A. flavus when colonizing peanut seeds. AF production in colonized seeds was decreased in the deletion mtfA strain and particularly in the overexpression strain, where only trace amounts were detected. Interestingly, a more rapid colonization of the seed tissue occurred when mtfA was overexpressed, coinciding with an increase in lipase activity and faster maceration of the oily part of the seed. Full article

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Open AccessFeature PaperArticle

Reduction of Aflatoxins in Apricot Kernels by Electronic and Manual Color Sorting

by Rosanna Zivoli, Lucia Gambacorta, Luca Piemontese and Michele Solfrizzo

Toxins 2016, 8(1), 26; https://doi.org/10.3390/toxins8010026 - 19 Jan 2016

Cited by 13 | Viewed by 6830

Abstract

The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched, peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) from healthy ones. [...] Read more.

The efficacy of color sorting on reducing aflatoxin levels in shelled apricot kernels was assessed. Naturally-contaminated kernels were submitted to an electronic optical sorter or blanched, peeled, and manually sorted to visually identify and sort discolored kernels (dark and spotted) from healthy ones. The samples obtained from the two sorting approaches were ground, homogenized, and analysed by HPLC-FLD for their aflatoxin content. A mass balance approach was used to measure the distribution of aflatoxins in the collected fractions. Aflatoxin B₁ and B₂ were identified and quantitated in all collected fractions at levels ranging from 1.7 to 22,451.5 µg/kg of AFB₁ + AFB₂, whereas AFG₁ and AFG₂ were not detected. Excellent results were obtained by manual sorting of peeled kernels since the removal of discolored kernels (2.6%–19.9% of total peeled kernels) removed 97.3%–99.5% of total aflatoxins. The combination of peeling and visual/manual separation of discolored kernels is a feasible strategy to remove 97%–99% of aflatoxins accumulated in naturally-contaminated samples. Electronic optical sorter gave highly variable results since the amount of AFB₁ + AFB₂ measured in rejected fractions (15%–18% of total kernels) ranged from 13% to 59% of total aflatoxins. An improved immunoaffinity-based HPLC-FLD method having low limits of detection for the four aflatoxins (0.01–0.05 µg/kg) was developed and used to monitor the occurrence of aflatoxins in 47 commercial products containing apricot kernels and/or almonds commercialized in Italy. Low aflatoxin levels were found in 38% of the tested samples and ranged from 0.06 to 1.50 μg/kg for AFB₁ and from 0.06 to 1.79 μg/kg for total aflatoxins. Full article

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Open AccessArticle

Hepatic Transcriptome Responses of Domesticated and Wild Turkey Embryos to Aflatoxin B₁

by Melissa S. Monson, Carol J. Cardona, Roger A. Coulombe and Kent M. Reed

Toxins 2016, 8(1), 16; https://doi.org/10.3390/toxins8010016 - 06 Jan 2016

Cited by 19 | Viewed by 5942

Abstract

The mycotoxin, aflatoxin B₁ (AFB₁) is a hepatotoxic, immunotoxic, and mutagenic contaminant of food and animal feeds. In poultry, AFB₁ can be maternally transferred to embryonated eggs, affecting development, viability and performance after hatch. Domesticated turkeys (Meleagris gallopavo [...] Read more.

The mycotoxin, aflatoxin B₁ (AFB₁) is a hepatotoxic, immunotoxic, and mutagenic contaminant of food and animal feeds. In poultry, AFB₁ can be maternally transferred to embryonated eggs, affecting development, viability and performance after hatch. Domesticated turkeys (Meleagris gallopavo) are especially sensitive to aflatoxicosis, while Eastern wild turkeys (M. g. silvestris) are likely more resistant. In ovo exposure provided a controlled AFB₁ challenge and comparison of domesticated and wild turkeys. Gene expression responses to AFB₁ in the embryonic hepatic transcriptome were examined using RNA-sequencing (RNA-seq). Eggs were injected with AFB₁ (1 μg) or sham control and dissected for liver tissue after 1 day or 5 days of exposure. Libraries from domesticated turkey (n = 24) and wild turkey (n = 15) produced 89.2 Gb of sequence. Approximately 670 M reads were mapped to a turkey gene set. Differential expression analysis identified 1535 significant genes with |log₂ fold change| ≥ 1.0 in at least one pair-wise comparison. AFB₁ effects were dependent on exposure time and turkey type, occurred more rapidly in domesticated turkeys, and led to notable up-regulation in cell cycle regulators, NRF2-mediated response genes and coagulation factors. Further investigation of NRF2-response genes may identify targets to improve poultry resistance. Full article

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Open AccessArticle

Identification and Quantification of a Toxigenic and Non-Toxigenic Aspergillus flavus Strain in Contaminated Maize Using Quantitative Real-Time PCR

by J. Erik Mylroie, Seval Ozkan, Renuka Shivaji, Gary L. Windham, Michael N. Alpe and W. Paul Williams

Toxins 2016, 8(1), 15; https://doi.org/10.3390/toxins8010015 - 04 Jan 2016

Cited by 10 | Viewed by 5835

Abstract

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective [...] Read more.

Aflatoxins, which are produced by Aspergillus flavus, are toxic to humans, livestock, and pets. The value of maize (Zea mays) grain is markedly reduced when contaminated with aflatoxin. Plant resistance and biological control using non-toxin producing strains are considered effective strategies for reducing aflatoxin accumulation in maize grain. Distinguishing between the toxin and non-toxin producing strains is important in determining the effectiveness of bio-control strategies and understanding inter-strain interactions. Using polymorphisms found in the fungal rRNA intergenic spacer region (IGS) between a toxigenic strain of A. flavus (NRRL 3357) and the non-toxigenic strain used in the biological control agent Afla-Guard^® (NRRL 21882), we developed a set of primers that allows for the identification and quantification of the two strains using quantitative PCR. This primer set has been used to screen maize grain that was inoculated with the two strains individually and co-inoculated with both strains, and it has been shown to be effective in both the identification and quantification of both strains. Screening of co-inoculated ears from multiple resistant and susceptible genotypic crosses revealed no significant differences in fungal biomass accumulation of either strain in the field tests from 2010 and 2011 when compared across the means of all genotypes. Only one genotype/year combination showed significant differences in strain accumulation. Aflatoxin accumulation analysis showed that, as expected, genotypes inoculated with the toxigenic strain accumulated more aflatoxin than when co-inoculated with both strains or inoculated with only the non-toxigenic strain. Furthermore, accumulation of toxigenic fungal mass was significantly correlated with aflatoxin accumulation while non-toxigenic fungal accumulation was not. This primer set will allow researchers to better determine how the two fungal strains compete on the maize ear and investigate the interaction between different maize lines and these A. flavus strains. Full article

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AFM₁ in Milk: Physical, Biological, and Prophylactic Methods to Mitigate Contamination

by Laura Giovati, Walter Magliani, Tecla Ciociola, Claudia Santinoli, Stefania Conti and Luciano Polonelli

Toxins 2015, 7(10), 4330-4349; https://doi.org/10.3390/toxins7104330 - 23 Oct 2015

Cited by 100 | Viewed by 10104

Abstract

Aflatoxins (AFs) are toxic, carcinogenic, immunosuppressive secondary metabolites produced by some Aspergillus species which colonize crops, including many dietary staple foods and feed components. AFB₁ is the prevalent and most toxic among AFs. In the liver, it is biotransformed into AFM₁ [...] Read more.

Aflatoxins (AFs) are toxic, carcinogenic, immunosuppressive secondary metabolites produced by some Aspergillus species which colonize crops, including many dietary staple foods and feed components. AFB₁ is the prevalent and most toxic among AFs. In the liver, it is biotransformed into AFM₁, which is then excreted into the milk of lactating mammals, including dairy animals. AFM₁ has been shown to be cause of both acute and chronic toxicoses. The presence of AFM₁ in milk and dairy products represents a worldwide concern since even small amounts of this metabolite may be of importance as long-term exposure is concerned. Contamination of milk may be mitigated either directly, decreasing the AFM₁ content in contaminated milk, or indirectly, decreasing AFB₁ contamination in the feed of dairy animals. Current strategies for AFM₁ mitigation include good agricultural practices in pre-harvest and post-harvest management of feed crops (including storage) and physical or chemical decontamination of feed and milk. However, no single strategy offers a complete solution to the issue. Full article

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Detoxification of Aflatoxin-Contaminated Maize by Neutral Electrolyzed Oxidizing Water

by Samantha Jardon-Xicotencatl, Roberto Díaz-Torres, Alicia Marroquín-Cardona, Tania Villarreal-Barajas and Abraham Méndez-Albores

Toxins 2015, 7(10), 4294-4314; https://doi.org/10.3390/toxins7104294 - 23 Oct 2015

Cited by 39 | Viewed by 8485

Abstract

Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human [...] Read more.

Aflatoxins, a group of extremely toxic mycotoxins produced by Aspergillus flavus, A. parasiticus and A. nomius, can occur as natural contaminants of certain agricultural commodities, particularly maize. These toxins have been shown to be hepatotoxic, carcinogenic, mutagenic and cause severe human and animal diseases. The effectiveness of neutral electrolyzed oxidizing water (NEW) on aflatoxin detoxification was investigated in HepG2 cells using several validation methodologies such as the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay, the induction of lipid peroxidation, the oxidative damage by means of glutathione modulation, the Ames test and the alkaline Comet assay. Our results showed that, after the aflatoxin-contaminated maize containing 360 ng/g was soaked in NEW (60 mg/L available chlorine, pH 7.01) during 15 min at room temperature, the aflatoxin content did not decrease as confirmed by the immunoaffinity column and ultra performance liquid chromatography methods. Aflatoxin fluorescence strength of detoxified samples was similar to untreated samples. However, aflatoxin-associated cytotoxicity and OPEN ACCESS Toxins 2015, 7 4295 genotoxicity effects were markedly reduced upon treatment. According to these results, NEW can be effectively used to detoxify aflatoxin-contaminated maize. Full article

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Extracellular Xylanolytic and Pectinolytic Hydrolase Production by Aspergillus flavus Isolates Contributes to Crop Invasion

by Jay E. Mellon

Toxins 2015, 7(8), 3257-3266; https://doi.org/10.3390/toxins7083257 - 19 Aug 2015

Cited by 10 | Viewed by 4531

Abstract

Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium [...] Read more.

Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents) and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase) P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates. Full article

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