Ratiometric electrochemical aptasensor based on split aptamer and Au-rGO for detection of aflatoxin M1.

A novel ratiometric electrochemical aptasensor based on split aptamer and Au-reduced graphene oxide (Au-rGO) nanomaterials was proposed to detect aflatoxin M1 (AFM1). In this work, Au-rGO nanomaterials were coated on the electrode through the electrodeposition method to increase the aptamer enrichment. We split the aptamer of AFM1 into 2 sequences (S1 and S2), where S1 was immobilized on the electrode due to the Au-S bond, and S2 was tagged with methylene blue (MB) and acted as a response signal. A complementary strand to S1 (CS1) labeled with ferrocene (Fc) was introduced as another reporter. In the presence of AFM1, CS1 was released from the electrode surface due to the formation of the S1-AFM1-S2 complex, leading to a decrease in Fc and an increase in the MB signal. The developed ratiometric aptasensor exhibited a linear range of 0.03 µg L-1 to 2.00 µg L-1, with a detection limit of 0.015 µg L-1 for AFM1 detection. The ratiometric aptasensor also showed a linear relationship from 0.2 µg L-1 to 1.00 µg L-1, with a detection limit of 0.05 µg L-1 in natural milk after sample pretreatment, indicating the successful application of the developed ratiometric aptasensor. Our proposed strategy provides a new way to construct aptasensors with high sensitivity and selectivity.

Omics analysis revealed the intestinal toxicity induced by aflatoxin B1 and aflatoxin M1.

Aflatoxin B1 (AFB1), a common mycotoxin, can occur in agricultural products. As a metabolite of AFB1, aflatoxin M1 (AFM1) mainly exist in dairy products. These two mycotoxins threaten human health, although it is unclear how they affect the function of the intestinal barrier. In this study, mice were exposed to AFB1 (0.3 mg/kg body b.w.) and AFM1(3.0 mg/kg b.w.) either individually or in combination for 28 days to explore the main differentially expressed proteins (DEPs) and the associated enriched pathways. These findings were preliminarily verified by the transcriptomic and proteomic analyses in differentiated Caco-2 cells. The results revealed that AFB1 and AFM1 exposure in mice disrupted the function of the intestinal barrier, and the combined toxicity was greater than that of each toxin alone. Further proteomic analysis in mice demonstrated that the mechanisms underlying these differences could be explained as follows: (i) lipid metabolism was enriched by AFB1-induced DEPs. (ii) protein export pathway was stimulated by AFM1-induced DEPs. (iii) cell metabolic ability was inhibited (as evidenced by changes in UDP-GT1, UDP-GT2, and Gatm6), apoptosis was induced (MAP4K3), and epithelial cell integrity was disrupted (Claudin7 and IQGAP2), resulting in more extensive intestinal damage after combined treatment. In conclusion, the hazardous impact of co-exposure to AFB1 and AFM1 from proteomic perspectives was demonstrated in the present study.

AFM1 exposure in male balb/c mice and intervention strategies against its immuno-physiological toxicity using clay mineral and lactic acid bacteria alone or in combination.

CONTEXT: Aflatoxins are the most harmful mycotoxins that cause human and animal health concerns. Aflatoxin M1 (AFM1) is the primary hydroxylated metabolite of aflatoxin B1 and is linked to the development of hepatocellular carcinoma and immunotoxicity in humans and animals. Because of the important role of dairy products in human life, especially children, AFM1 is such a major concern to humans because of its frequent occurrence in dairy products at concentrations high enough to cause adverse effects to human and animal health. Reduced its bioavailability becomes a high priority in order to protect human and animal health. OBJECTIVES: This study aimed to investigate, in vivo, the ability of lactic acid bacteria (lactobacillus rhamnosus GAF01, LR) and clay mineral (bentonite, BT) mixture to mitigate/reduce AFM1-induced immunotoxicity, hepatotoxicity, nephrotoxicity and oxidative stress in exposed Balb/c mice. MATERIALS AND METHODS: The in vivo study was conducted using male Balb/c mice that treated, orally, by AFM1 alone or in combination with LR and/or BT, daily for 10 days as follows: group 1 control received 200 µl of PBS, group 2 treated with LR alone (2.108 CFU/mL), group 3 treated with BT alone (1 g/kg bw), group 4 treated with AFM1 alone (100 µg/kg), group 5 co-treated with LR + AFM1, group 6 co-treated with BT + AFM1, group 7 co-treated with BT + LR + AFM1. Forty-eight h after the end of the treatment, the mice were sacrificed and the blood, spleen, thymus, liver and kidney were collected. The blood was used for biochemical and immunological study. Spleen and thymus samples were used to thymocytes and splenocytes assessments. Liver and kidney samples were the target for evaluation of oxidative stress enzymes status and for histological assays. RESULTS: The results showed that AFM1 caused toxicities in male Blab/c mice at different levels. Treatment with AFM1 resulted in severe stress of liver and kidney organs indicated by a significant change in the biochemical and immunological parameters, histopathology as well as a disorder in the profile of oxidative stress enzymes levels. Also, it was demonstrated that AFM1 caused toxicities in thymus and spleen organs. The co-treatment with LR and/or BT significantly improved the hepatic and renal tissues, regulated antioxidant enzyme activities, spleen and thymus viability and biochemical and immunological parameters. LR and BT alone showed to be safe during the treatment. CONCLUSION: In summary, the LR and/or BT was able to reduce the biochemical, histopathological and immunological damages induced by AFM1 and indeed it could be exploited as one of the biological strategies for food and feedstuffs detoxification.

Aflatoxin M1 Concentrations, Adulterants, Microbial Loads, and Physicochemical Properties of Raw Milk Collected From Nekemte City, Ethiopia.

Milk is an essential part of the human diet and is a nutrient-rich food that improves nutrition and food security. The aim of this study was to determine the presence and concentration of aflatoxin M1 (AFM1), adulterants, microbial loads, and physicochemical properties of raw cow's milk (CM) in Nekemte City, Ethiopia. A total of 12 samples of fresh CM were purposefully collected from four kebeles in the city (Bake Jama, Burka Jato, Cheleleki, and Bakanisa Kese) based on the potential of each milk production and distributor site. The AFM1 concentration was determined by high-performance liquid chromatography (HPLC) with a Sigma-Aldrich standard (St. Louis, MO, USA). The concentrations of AFM1 in Bake Jama, Burka Jato, Cheleleki, and Bakanisa Kese were found to be 0.01-0.03 g/L, 0.31-0.35 g/L, 0.19-0.21 g/L, and 0.04-0.07 g/L, respectively. The concentrations of AFM1 in the present study varied significantly (p < 0.05) and ranged from 0.01 g/L to 0.35 g/L. These results show that of the 12 samples tested, all were positive for AFM1 and contaminated to varying degrees. The results of this study also revealed that the concentration of AFM1 in 7 (58%) of the 12 milk samples was above the European Union's (EU) maximum tolerance limit (0.05 g/L). The present study also revealed that of the investigated adulterants, only the addition of water had positive effects on three milk samples, while the remaining adulterants were not detected in any of the milk samples. The total bacterial count (TBC) and total coliform count (TCC) were significantly (p < 0.05) different and ranged from 5.53 to 6.82 log10cfumL-1 and from 4.21 to 4.74 log10cfumL-1, respectively. The physicochemical properties of the milk samples in the present study were significantly (p < 0.05) different and ranged from 2.8% to 5.75% fat, 7.03% to 9.75% solid-not-fat (SNF), 2.35% to 3.61% protein, 3.33% to 5.15% lactose, 11.54% to 13.69% total solid, 0.16% to 0.18% titratable acid, 26.7 to 32.1°C, 6.35 to 6.55 pH, and 1.027 to 1.030 specific gravity. The physicochemical parameters of the raw milk in the study area met the required quality standards. Hence, further studies are required to determine the extent of the problem and the factors associated with high levels of AFM1 in raw milk in the study areas, including the detection of aflatoxin B1 (AFB1) in animal feed.

Effects of chronic low-dose aflatoxin B1 exposure in lactating Florida dairy goats.

In the past few years there has been a growing trend in the prevalence of aflatoxins, attributable to climate change, in substances destined for animal feeding, together with an increase in dairy product consumption. These facts have triggered great concern in the scientific community over milk pollution by aflatoxin M1. Therefore, our study aimed to determine the transfer of aflatoxin B1 from the diet into milk as AFM1 in goats exposed to different concentrations of AFB1, and its possible effect on the production and serological parameters of this species. For this purpose, 18 goats in late lactation were divided into 3 groups (n = 6) and exposed to different daily doses of aflatoxin B1 (T1 = 120 µg; T2 = 60 µg, and control = 0 µg), during 31 d. Pure aflatoxin B1 was administered 6 h before each milking in an artificially contaminated pellet. The milk samples were taken individually in sequential samples. Milk yield and feed intake were recorded daily, and a blood sample was extracted on the last day of exposure. No aflatoxin M1 was detected, either in the samples taken before the first administration, or in the control group ones. The aflatoxin M1 concentration detected in the milk (T1 = 0.075 µg/kg; T2 = 0.035 µg/kg) increased significantly on a par with the amount of aflatoxin B1 ingested. The amount of aflatoxin B1 ingested did not have any influence on aflatoxin M1 carryover (T1 = 0.066% and T2 = 0.060%), these being considerably lower than those described in dairy goats. Thus, we concluded that the concentration of aflatoxin M1 in milk follows a linear relationship with respect to the aflatoxin B1 ingested, and that the aflatoxin M1 carryover was not affected by the administration of different aflatoxin B1 doses. Similarly, no significant changes in the production parameters after chronic exposure to aflatoxin B1 were observed, revealing a certain resistance of the goat to the possible effects of that aflatoxin.

A network meta-analysis on the efficacy of different mycotoxin binders to reduce aflatoxin M1 in milk after aflatoxin B1 challenge in dairy cows.

The objective of this network meta-analysis was to determine the efficacy of different mycotoxin binders (MTB) to reduce aflatoxin M1 (AFM1) in milk. A literature search was conducted to identify in vivo research papers from different databases. Inclusion criteria were in vivo, dairy cows, description of the MTB used, doses of MTB, aflatoxin inclusion in the diet, and concentration of AFM1 in milk. Twenty-eight papers with 131 data points were selected. Binders used in the studies were hydrated sodium calcium aluminosilicate (HSCAS), yeast cell wall (YCW), bentonite, and mixes of several MTB (MX). The response variables were AFM1 concentration, AFM1 reduction in milk, total AFM1 excreted in milk, and transfer of aflatoxin from feed to AFM1 in milk. Data were analyzed with CINeMA and GLIMMIX procedures with the WEIGHT statement of SAS (SAS Inst. Inc.). The AFM1 concentration in milk decreased for bentonite (0.3 µg/L ± 0.05; mean ± SE) and HSCAS (0.4 µg/L ± 0.12), and tended to decrease for MX (0.6 µg/L ± 0.13) but was similar for YCW (0.6 µg/L ± 0.12), compared with control (0.7 µg/L ± 0.12). The percentage reduction of AFM1 in milk was similar for all MTB and different from control with a range of reduction from 25% for YCW to 40% for bentonite. The excretion of AFM1 in milk was lower in YCW (5.3 µg/L ± 2.37), HSCAS (13.8 µg/L ± 3.31), and MX (17.1 µg/L ± 5.64), and not affected by bentonite (16.8 µg/L ± 3.33) compared with control (22.1 µg/L ± 5.33). The transfer of aflatoxin B1 from feed into AFM1 in milk was lowest in bentonite (0.6% ± 0.12), MX (1.04% ± 0.27), and HSCAS (1.04% ± 0.21), and not affected in YCW (1.4% ± 0.10), compared with control (1.7% ± 0.35). The meta-analysis results indicate that all MTB reduced the AFM1 transfer into milk, where bentonite had the highest capacity and YCW the lowest.

The coexistence of aflatoxin M1 and ochratoxin A induced intestinal barrier disruption via the regulation of key differentially expressed microRNAs and long non-coding RNAs in BALB/c mice.

Food safety can be seriously threatened by the existence of both aflatoxin M1 (AFM1) and ochratoxin A (OTA) in milk and corresponding products. The importance of intestine integrity in preserving human health is widely understood in vitro, but the fundamental processes by which AFM1 and OTA cause disruption of the intestinal barrier are as yet unknown, especially in vivo. Based on the analysis of the whole transcriptome of BALB/c mice, the competing endogenous RNA (ceRNA) regulation network was obtained in the current study. Each of 12 mice were separated into five treatments: saline solution treatment, 1.0% DMSO vehicle control treatment, 3.0 mg/kg b.w. individual AFM1 treatment (AFM1), 3.0 mg/kg b.w. individual OTA treatment (OTA), and combined mycotoxins treatment (AFM1 +OTA). The study period lasted 28 days. The jejunum tissue was collected for the histological assessment and whole transcriptome analysis, and the whole blood was collected, and determination of serum biochemical indicators. The phenotypic results demonstrated that AFM1 and OTA caused intestinal barrier disruption via an increased apoptosis level and decreased expression of tight junction (TJ) proteins. The ceRNA network demonstrated that AFM1 and OTA induced cell apoptosis through activating the expression of DUSP9 and suppressing the expression of PLA2G2D, which were regulated by differentially expressed microRNAs (DEmiRNAs) (miR-124-y, miR-194-z, miR-224-x, and miR-452-x) and differentially expressed long non-coding RNAs (DElncRNAs) (FUT8 and GPR31C). And AFM1 and OTA decreased TJ proteins via inhibiting the expression of PAK6, which was regulated by several important DEmiRNAs and DElncRNAs. These DE RNAs in intestinal integrity were involved in MAPK and Ras signaling pathway. Overall, our findings expand the current knowledge regarding the potential mechanisms of intestinal integrity disruption brought on by AFM1 and OTA in vivo.

A novel electrochemical aptasensor based on layer-by-layer assembly of DNA-Au@Ag conjugates for rapid detection of aflatoxin M1 in milk samples.

Aflatoxin M1 (AFM1) is a common toxin in dairy products that causes acute and chronic human health disorders. Thus, the development of a rapid and accurate AFM1 detection method is of vital importance for food safety monitoring. This work was to develop a novel electrochemical aptasensor for sensitive and specific determination of AFM1. The dendritic-like nanostructure was formed on the gold electrode surface by layer-by-layer assembly of gold-silver core-shell nanoparticles modified with DNA conjugates. In the presence of AFM1, the specific recognition between AFM1 and Apt caused the disassociation of the DNA controlled dual Au@Ag conjugates from the surface of the electrode, causing less methylene blue to bind to the surface and weakening the electrochemical signal. The more AFM1 there is, the weaker the electrochemical signal. Transmission electron microscope results showed that the successfully synthesized Au@Ag nanoparticles exhibited a core-shell structure with Au as core and Ag as shell, and their average diameter was about 30 nm. Under optimal conditions, the electrochemical aptasensor showed a wide detection ranging from 0.05 ng mL-1 to 200 ng mL-1, and a low detection limit of 0.02 ng mL-1. Moreover, the proposed strategy has been successfully applied to the detection of AFM1 in cow, goat, and sheep milk samples with satisfactory recoveries ranging from 91.10% to 104.05%. This work can provide a novel rapid detection method for AFM1, and also provide a new sensing platform for the detection of other toxins.

A Strategy for Sample Preparation: Using Egg White Gel to Promote the Determination of Aflatoxin M1 Content in Milk Samples.

The analysis of food samples is a challenging task. The high complexity of food matrices hinders the extraction and detection of analytes from them. Therefore, the correct preparation of food samples is a crucial step for their subsequent analysis, as it achieves the proper isolation and preconcentration of analytes and removes the interfering proportion of the food matrix before instrumental analysis. We aimed to develop a method that not only satisfies the requirement of detecting trace compounds in complex matrices but also achieves a "greener" approach by reducing the use of organic solvents and non-degradable materials to minimize the health hazards posed to the operators as well as pollution to the environment. In this study, we prepared egg white as a concentrated gel and used this material for the biological purification of milk samples. After the milk protein was removed by acidification and salting, the residual amount of aflatoxin M1 in milk samples was quantitatively determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the novel egg white purification method possessed advantages over the immunoaffinity technique used as the reference method in extraction recovery, sensitivity, repeatability, and operability. The limit of detection (LOD) was 0.001 µg/kg. In spiked samples containing 0.01 µg/kg to 2 µg/kg of AFM1, the average recovery was 88.3-94.7%, with a precision of 6.1-11.0%. Improved repeatability was obtained by significantly reducing the operation time and resource requirements compared with the immunoaffinity technique currently used internationally. This study provides a reference for the further improvement of the relevant international standards in place for the detection of aflatoxin M1 in milk.

Influence of season and lactational stage on aflatoxin M1 and ochratoxin A in human milk in a cohort study from southeastern region of Turkey.

The aim was to evaluate the changes in aflatoxin M1 (AFM1) and ochratoxin A (OTA) levels in human breast milk (HBM) during the first five postpartum months according to the sampling season in a cohort study from Sanliurfa. From 78 healthy lactating mothers, HBM was taken at the 5-14 days postpartum (D5-14) and the 6th and 18th weeks postpartum (W6 and W18). Mycotoxin levels were analyzed with competitive ELISA. Generalized Estimating Equations with repeated measures (three-correlation matrix dimension) revealed a significantly higher mean AFM1 level at W6 than that on D5-14. AFM1 and OTA levels in winter and spring were considerably higher than that in summer and autumn. Maternal smoke exposure, body mass index, history of moldy food exposure, birth order, and breastfeeding type did not influence the results. Whilst season had a marked effect on the milk levels of both analytes, lactation stage affected AFM1 more notable than OTA.

Association between aflatoxin M1 excretion in milk and indicators of rumen fermentation in bovines.

Aflatoxins and its metabolites negatively impact the ruminant health and production. The present cross-sectional study was aimed to determine the effect of aflatoxins on rumen fermentation by deducing the correlation between the aflatoxin M1 (AFM1) excretion in milk and indicators of rumen fermentation in bovines. The indicators of rumen fermentation were taken into account and correlated with AFM1 concentration in milk of 120 bovines (cattle (n = 82) and buffalo (n = 38)). The AFM1 in milk samples (n = 120) was quantified by ELISA kit. The correlation analysis revealed that with increase in excretion of AFM1 in milk, the pH (r = 0.38), methylene blue reduction time (MBRT) (r = 0.43), sedimentation activity time (SAT) (r = 0.31) and ammonia nitrogen content (r = 0.34) of rumen liquor increase, whereas the total volatile fatty acid (TVFA) content (r = - 0.25), total bacterial count (TBC) (r = - 0.43) and total protozoal count (TPC) (r = - 0.14) of rumen liquor decrease. The results of the present study suggest that the presence of aflatoxins in rumen could have negative effect on the process of rumen fermentation. Therefore, the prevention of primary entry point(s) of AFB1 through the feed of bovines is important for the animal health as well as public health.

[AFM1 Content Survey in Dairy Products for Infants].

Infant formula in liquid for childcare can be stored at room temperature for a certain period of time, reducing the burden of childcare and preparing for disasters. Against this background, domestic manufacturing and sales began in March 2019. AFM1 is a metabolite of aflatoxin B1 (AFB1), a carcinogenic mycotoxin, and is contained in the milk of livestock fed a diet contaminated with AFB1. At present, standard values have not been set for infant formula in liquid as well as prepared infant formula in liquid, and infants consume a large amount of dairy products per body weight, so care must be taken in the intake.In this study, we investigated the actual condition of AFM1 content in dairy products with high intake of infants. As a result of the investigation, the AFM1 of the detected dairy products was 0.001 to 0.005 µg/kg, which was extremely small compared to the AFM1 in the dairy products reported so far. Since infant nutrition depends on dairy products, it is undeniable that they may consume more than adults, so continuous research is needed.

Occurrence and exposure assessment of Aflatoxin M1 in milk and milk products in India.

Milk containing Aflatoxin M1 (AFM1) poses a serious health risk to consumers. Present study was undertaken to determine levels of AFM1 in 146 milk and value added dairy products sold in retail markets of Chhattisgarh, India using HPLC coupled with fluorescence detector. A total of 52 samples (35.6%) were found to contain AFM1 with overall concentrations ranging from nd - 2.608 µg/L. The contamination levels were higher in non-fermented milk products than fermented milk products samples, although this difference was statistically non-significant (p > 0.05). AFM1 concentrations above maximum permissible limits established by the European Commission were found in 94.2% of positive samples. Health risk assessments ascertained that the estimated daily intakes for AFM1 is higher than the established tolerable daily intakes for both adults and children (Hazard Index > 1), there by implying a potentially high risk to consumer's health. Current investigation provides valuable information regarding contamination of raw as well as value added milk products sold in Indian markets. Therefore, to protect consumer's health and promote dairy trade; there is an urgent need to increase farmer's knowledge on good storage practices of feed and fodder. Further, stringent enforcement of food safety regulations is imperative to safeguard and promote human health.

Aflatoxin M1 in Africa: Exposure Assessment, Regulations, and Prevention Strategies - A Review.

Aflatoxins are the most harmful mycotoxins causing health problems to human and animal. Many acute aflatoxin outbreaks have been reported in Africa, especially in Kenya and Tanzania. When ingested, aflatoxin B1 is converted by hydroxylation in the liver into aflatoxin M1, which is excreted in milk of dairy females and in urine of exposed populations. This review aims to highlight the AFM1 studies carried out in African regions (North Africa, East Africa, West Africa, Central Africa, and Southern Africa), particularly AFM1 occurrence in milk and dairy products, and in human biological fluids (breast milk, serum, and urine) of the populations exposed. Strategies for AFM1 detoxification will be considered, as well as AFM1 regulations as compared to the legislation adopted worldwide and the assessment of AFM1 exposure of some African populations. Egypt, Kenya, and Nigeria have the highest number of investigations on AFM1 in the continent. Indeed, some reports showed that 100% of the samples analyzed exceeded the EU regulations (50 ng/kg), especially in Zimbabwe, Nigeria, Sudan, and Egypt. Furthermore, AFM1 levels up to 8,000, 6,999, 6,900, and 2040 ng/kg have been reported in milk from Egypt, Kenya, Sudan, and Nigeria, respectively. Data on AFM1 occurrence in human biological fluids have also shown that exposure of African populations is mainly due to milk intake and breastfeeding, with 85-100% of children being exposed to high levels. Food fermentation in Africa has been tried for AFM1 detoxification strategies. Few African countries have set regulations for AFM1 in milk and derivatives, generally similar to those of the Codex alimentarius, the US or the EU standards.

Transcriptomics and flow cytometry reveals the cytotoxicity of aflatoxin B1 and aflatoxin M1 in bovine mammary epithelial cells.

Aflatoxin is a known mycotoxin that pollutes various grains widely in the environment. Aflatoxin B1 (AFB1) and Aflatoxin M1 (AFM1) have been shown to induce cytotoxicity in many cells, yet their effects on mammary epithelial cells remain unclear. In this study, we examined the toxicity and the effects of AFB1 and AFM1 on bovine mammary epithelial cells (BME cells). The cells were treated with AFB1 or AFM1 at a concentration of 0-10 mg/L for 24 or 48 h, followed by cytotoxicity assays, flow cytometry, and transcriptomics. Our results demonstrated that AFB1 and AFM1 induced cell proliferation inhibition, apoptosis and cell cycle arrest. However, the level of intracellular reactive oxygen species has no significant difference. The RNA-Seq results also showed that AFB1 and AFM1 changed many related gene expressions like apoptosis and oxidative stress, cycle, junction, and signaling pathway. Taken together, AFB1 and AFM1 were found to affect cytotoxicity and related gene changes in BME cells. Notably, this study reported that 2 mg/L of AFB1 and AFM1 affected the expression of methylation-related genes, and ultimately altered the rate of m6A methylation in RNA. It may provide a potential direction for toxins to indirectly regulate gene expression by affecting RNA methylation modification. Our research provides some novel insights and data about AFB1 and AFM1 toxicity in BME cells.

miR-155 influences cell-mediated immunity in Balb/c mice treated with aflatoxin M1.

Aflatoxin M1 (AFM1) is a 4-hydroxylated metabolite of aflatoxin B1 (AFB1). It induces various toxicological effects including immunotoxicity. In the present study, we investigated the effects of AFM1 on immune system and its modulation by MicroRNA (miR)-155. AFM1 was administered intraperitoneally at doses of 25 and 50 µg/kg for 28 days to Balb/c mice and different immune system parameters were analyzed. The levels of miR-155 and targeted proteins were evaluated in isolated T cells from spleens of mice. Spleen weight was reduced in mice exposed to AFM1 compared to negative control. Proliferation of splenocytes in response to phytohemagglutinin-A was reduced in mice exposed to AFM1. IFN-γ was decreased in mice exposed to AFM1, whereas IL-10 was increased. Concentration of IL-4 did not change different in mice exposed to AFM1 compared to negative control. Exposure to AFM1 reduced the expression of miR-155. Significant upregulation of phosphatidylinositol-3, 4, 5-trisphosphate 5-phosphatase 1 (Ship1) and suppressor of cytokine signaling 1 (Socs1) was observed in isolated T cells from spleens of mice treated with AFM1, but the transcription factor Maf (c-MAF) was not affected. These results suggest that miR-155 and targeted proteins might be involved in the immunotoxicity observed in mice exposed to AFM1.

An ultrasensitive, homogeneous fluorescence quenching immunoassay integrating separation and detection of aflatoxin M1 based on magnetic graphene composites.

A homogeneous fluorescence quenching immunoassay is described for simultaneous separation and detection of aflatoxin M1 (AFM1) in milk. The novel assay relies on monoclonal antibody (mAb) functionalized Fe3O4 decorated reduced-graphene oxide (rGO-Fe3O4-mAb) as both capture probe and energy acceptor, combined with tetramethylrhodamine cadaverine-labeled aflatoxin B1 (AFB1-TRCA) as the energy donor. In the assay, AFB1-TRCA binds to rGO-Fe3O4-mAb in the absence of AFM1, quenching the fluorescence of TRCA by resonance energy transfer. Significantly, the immunoassay integrates sample preparation and detection into a single step, by using magnetic graphene composites to avoid washing and centrifugation steps, and the assay can be completed within 10 min. Under optimized conditions, the visual and quantitative detection limits of the assay for AFM1 were 50 and 3.8 ng L-1, respectively, which were significantly lower than those obtained by fluorescence polarization immunoassay using the same immunoreagents. Owing to its operation and highly sensitivity, the proposed assay provides a powerful tool for the detection of AFM1.

A Review of the Use of Carbon Nanotubes and Graphene-Based Sensors for the Detection of Aflatoxin M1 Compounds in Milk.

This paper presents a comprehensive review of the detection of aflatoxin compounds using carbon allotrope-based sensors. Although aflatoxin M1 and its derivative aflatoxin B1 compounds have been primarily found in milk and other food products, their presence above a threshold concentration causes disastrous health-related anomalies in human beings, such as growth impairment, underweight and even carcinogenic and immunosuppressive effects. Among the many sensors developed to detect the presence of these compounds, the employment of certain carbon allotropes, such as carbon nanotubes (CNTs) and graphene, has been highly preferred due to their enhanced electromechanical properties. These conductive nanomaterials have shown excellent quantitative performance in terms of sensitivity and selectivity for the chosen aflatoxin compounds. This paper elucidates some of the significant examples of the CNTs and graphene-based sensors measuring Aflatoxin M1 (ATM1) and Aflatoxin B1 (AFB1) compounds at low concentrations. The fabrication technique and performance of each of the sensors are shown here, as well as some of the challenges existing with the current sensors.

The Protective Effects of Lactoferrin on Aflatoxin M1-Induced Compromised Intestinal Integrity.

Aflatoxin M1 (AFM1), the only toxin with maximum residue levels in milk, has adverse effects on the intestinal barrier, resulting in intestinal inflammatory disease. Lactoferrin (LF), one of the important bioactive proteins in milk, performs multiple biological functions, but knowledge of the protective effects of LF on the compromised intestinal barrier induced by AFM1 has not been investigated. In the present study, results using Balb/C mice and differentiated Caco-2 cells showed that LF intervention decreased AFM1-induced increased intestinal permeability, improved the protein expression of claudin-3, occludin and ZO-1, and repaired the injured intestinal barrier. The transcriptome and proteome were used to clarify the underlying mechanisms. It was found that LF reduced the intestinal barrier dysfunction caused by AFM1 and was associated with intestinal cell survival related pathways, such as cell cycle, apoptosis and MAPK signaling pathway and intestinal integrity related pathways including endocytosis, tight junction, adherens junction and gap junction. The cross-omics analysis suggested that insulin receptor (INSR), cytoplasmic FMR1 interacting protein 2 (CYFIP2), dedicator of cytokinesis 1 (DOCK1) and ribonucleotide reductase regulatory subunit M2 (RRM2) were the potential key regulators as LF repaired the compromised intestinal barrier. These findings indicated that LF may be an alternative treatment for the compromised intestinal barrier induced by AFM1.

Prevalence of contamination of aflatoxin M1 in milk: a retrospective analysis of studies conducted in Pakistan.

Aflatoxins, produced by multiple fungal species, are present in several kinds of food items and animal feed. Several studies conducted in Pakistan have reported the presence of aflatoxin M1 (AFM1) in milk. Hence, owing to the public health concern and absence of general statistics regarding the prevalence of AFM1 contamination, current study was aimed to investigate the prevalence of AFM1 in milk in Pakistan. For this study, various databases were searched from 2007 to 2020. A random effect model was applied for analytical purpose and heterogeneity of selected studies was investigated with an I2 index. Comprehensive meta-analysis (version 3) was used for analysis of data. According to the results, prevalence of AFM1 in milk was 84.4% (95% CI 75.0-90.7%). Regarding the heterogeneity based on meta-regression, it has been observed that there was a significant difference between the effect of year of study and sample size with prevalence of AFM1 in animal milk. These results suggest that AFM1 contamination in animal milk is high in Pakistan. Hence, continuous monitoring of AFM1 in animal milk requires utmost attention from the respective food and drug regulatory authorities of Pakistan so that the strict actions and preventive measures should be taken to prevent the prevalence of exposure of AFM1 in animal milk.