Study protocol to assess aflatoxin M1 health risks versus benefits of dairy consumption in Ethiopian children: an epidemiological trial and risk-benefit analysis.

INTRODUCTION: In Sidama, Ethiopia, animal-source foods can be difficult to access. Milk has important nutrients for child growth, but carries the risk of aflatoxin M1 (AFM1) contamination. AFM1 is a metabolite of the mycotoxin aflatoxin B1 (AFB1) in dairy feed; cows secrete AFM1 in milk when their feed contains AFB1 produced by Aspergillus fungi in maize, nuts and oilseeds. It is unknown whether AFM1 compromises child growth and health. METHODS AND ANALYSIS: This protocol paper describes our study in Sidama to determine the impact of milk consumption and AFM1 on child growth in the first 18 months of life. We will collect baseline and end-line data on dairy production, socioeconomic and nutritional factors of 1000 dairy-owning households with children ages 6-18 months at baseline; and gather samples of milk and dairy feed and child anthropometrics. We will conduct phone interviews every 6 months to ascertain changes in practices or child health. Dairy feed will be tested for AFB1; milk for AFM1, pathogens and nutrients. Controlling for herd size, socioeconomic, nutritional and behavioural factors, we will determine the association between child anthropometrics and milk consumption, as well as AFM1 exposure. We will examine whether AFM1 exposure affects child growth in the first 18 months of life, and weigh the benefits and risks of milk consumption. ETHICS AND DISSEMINATION: The protocol is approved by the Institutional Review Boards of the Ethiopian Public Health Institute (EPHI-IRB-481-2022), Michigan State University (STUDY00007996) and International Food Policy Research Institute (DSGD-23-0102). Written informed consent will be obtained from all participants, who may withdraw from the study at any time. Confidentiality of collected data will be given high priority during each stage of data handling. The study's findings will be disseminated through stakeholder workshops, local and international conferences, journal articles and technical reports.

Dietary Exposure and Risk Assessment of Multi-Mycotoxins (AFB1, AFM1, OTA, OTB, DON, T-2 and HT-2) in the Lebanese Food Basket Consumed by Adults: Findings from the Updated Lebanese National Consumption Survey through a Total Diet Study Approach.

Mycotoxins have been linked to adverse health impacts, including liver cancer and kidney diseases. The objectives of the current study were to evaluate the dietary exposure of Lebanese adults to multi-mycotoxins (aflatoxin B1 (AFB1), aflatoxin M1 (AFM1), ochratoxin A (OTA), ochratoxin B (OTB), deoxynivalenol (DON), T-2 and HT-2) and to assess their associated health risks. Hence, a nationally representative sample of 449 participants aged 18-64 years old were interviewed to obtain their socio-demographic characteristics, food consumption data and exposure estimates. A food frequency questionnaire and 24 h-recall were used to collect data. The concentration of mycotoxins in all foods consumed by the participants was collected from previous national published studies. The estimated daily intake (EDI), the hazard quotient (HQ) and the margin of exposure (MOE) were calculated. The total exposure to AFB1, AFM1, OTA and DON was 1.26, 0.39, 4.10 and 411.18 ng/kg bw/day, respectively. The MOE to AFB1, AFM1, OTA and DON in the Lebanese food basket was 316, 1454, 3539 and 510, respectively, indicating high health-related risks. Per food items, the MOE to AFB1 was below 10,000 in cereals (466.5), mainly in rice (827.9) and Burgul (4868.5). Similarly, the MOE to OTA in cereals was 1439, in which bread (4022), rice (7589) and bulgur (7628) were considered unsafe. Moreover, the MOE to DON in cereals (605) is alarming, especially in bread (632) and manakesh (6879). The MOE to AFM1 in dairy products was 1454, indicating health-related risks with a focus on yogurt (9788) and labneh (8153). As for the herbs/spices group and traditional dishes, the MOE to AFB1 was relatively lower than 10,000 (3690 and 1625, respectively), with a focus on thyme (2624) and kishik (3297), respectively. It is noteworthy that the MOE to DON and the MOE to OTA in traditional foods and coffee were lower than 10,000 (8047 and 8867, respectively). All hazard quotient (HQ) values were below 1, except the HQ value of milk and dairy products (1.96). The intake of some food groups varied between age categories, corresponding to differences in EDI between them. Thus, it is essential to put control measures in place to decrease the contamination and exposure to mycotoxins by Lebanese consumers.

Assessment of some chemical residues in Egyptian raw milk and traditional cheese.

Background: The assessment of risks related to food safety is becoming a challenge in developing countries with its consequent health hazards. Chemical risk assessment in dairy products is important to maintain consumer health locally and internationally. Since milk and dairy products are essential foods for a wide range of customers, mostly children, patients, and pregnant women, it is very important to estimate the risks of some chemical residues, such as pesticides, some heavy metals, and aflatoxins. Aim: This work aims to determine the levels of chemical contamination in milk and traditional Egyptian cheese. Methods: Heavy metals were determined in samples by atomic absorption spectrometry. GC-mass spectrometry (MS)/MS and LC-MS/MS were also used for measuring pesticide residues. The Aflatoxin M1 was determined by enzyme-linked immune-sorbent assay. Results: Raw milk samples were tested and showed elevated concentrations of lead and cadmium, (46% and 4%, respectively). The heavy metals detected in the Egyptian cheese samples were variable depending on the type of cheese. Moreover, p.p.-DDE phenofose was present in 45% and 29% of raw milk and Ras cheese samples, respectively. For Aflatoxin M1, only 7% of milk samples and 2.9% of Ras cheese samples exceeded the acceptable limits. Conclusion: More surveying and risk assessment of chemical residues in milk and milk products are essential for controlling health risks to consumers.

Occurrence of Aflatoxin M1 and Estimate of Dietary Exposure in Cheeses from Organic and Conventional Production Systems.

This study aimed to compare AFM1 occurrence in different cheese types produced by organic and conventional systems; and to evaluate the risk of food exposure to AFM1. A total of 176 commercial cheeses of 17 types were analyzed, 84 of organic and 92 of conventional production. Determination of AFM1 was performed by high- performance liquid chromatography (HPLC), being detected in 30.5% of samples, with 4.8% of organic cheese samples presenting quantifiable AFM1 values between 0.88 and 1.50 µg/kg. On the other hand, 4.3% of conventional cheese samples with values between 0.79 and 6.70 µg/kg. Two conventional cheese samples were above the limit of AFM1 allowed for cheeses by the Brazilian legislation. No statistical difference were found between organic and conventional cheeses regarding the occurrence (p = 0.1780) and concentration of AFM1 (p = 0.1810), according to the Chi-square and the T test, respectively. Estimated daily intake (EDI) and hazard index (HI) of dietary exposure to AFM1 were 0.26 ng/kg/day and 1.28 ng/kg/day, respectively, for conventional cheese samples, and 0.09 ng/kg/day and 0.47 ng/kg/day for organic samples, with no statistical difference for EDI (p = 0.1729) and HI (p = 0.1802) between the two production systems. Comparison between several cheese types from conventional and organic systems indicated that AFM1 is an obstacle to dairy production. Control and prevention of AFM1 contamination, as well as detoxification methods in the final products, are necessary. In the case of organic products, additional research is needed in order to determine which control and detoxification methods should be allowed in this production system.

Risk of exposure to aflatoxin M1 through consumption of cow's milk among children in Magadu, Morogoro.

Aflatoxin M1 (AFM1) contamination of milk affects the general population with particular attention to children who frequently consume milk as part of complementary food. This study determined AFM1 contamination of cow's milk and estimated the health risk of dietary AFM1 through consumption of cow's milk among children (6 to 36 months) in the Magadu ward of Morogoro region in Tanzania. A total of 165 mother-baby pairs were recruited and interviewed on child feeding practices with a focus on feeding of cow's milk in the past 24 h. Alongside the interview, 100 raw cows' milk samples were collected from subsampled respondent households and were analyzed for AFM1 using enzyme-linked immunosorbent assay (ELISA). The results showed that about 35% of the surveyed children consumed cow's milk in the form of plain milk, incorporated in porridge and/or tea. The amount consumed varied from 62.5 to 500 mls with a median of 125 (125, 250) mls at a frequency of 1 to 2 times a day. All raw cows' milk (100%) samples (n = 100) were found contaminated with AFM1 at concentrations ranging from 0.052 to 9.310 µg/L and median of 2.076 µg/L (1.27, 2.48). All samples were contaminated by AFM1 at levels above the limits of 0.05 µg/L of raw milk set by the Tanzania Bureau of Standard and the European Union, while 97% exceeded 0.5 µg/L set by the US Food and Drug Administration. Exposure to AFM1 due to consumption of cow's milk ranged from 0.0024 to 0.077 µg/kg bw per day with a median of 0.019 (0.0016, 0.026) µg/kg bw per day, while the margin of exposure (MOE) ranged from 5.19 to 166.76 and median 20.68 (15.33, 25.40) implying high risk of public health concern. This study recommends that advocacy on consumption of cows' milk to combat undernutrition in children should consider a holistic approach that considers the milk's safety aspect.

CRISPR-Cas12a-based colorimetric aptasensor for aflatoxin M1 detection based on oxidase-mimicking activity of flower-like MnO2 nanozymes.

Given the highly mutagenic and carcinogenic nature of Aflatoxin M1 (AFM1), the quantity assessment of AFM1 residues in milk and dairy products is necessary to maintain consumer health and food safety. Herein, CRISPR-Cas12a-based colorimetric aptasensor was developed using the catalytic activity of flower-like nanozymes of MnO2 and trans-cleavage property of CRISPR-Cas12a system to quantitatively detect AFM1. The basis of the developed colorimetric aptasensor relies on whether or not the CRISPR-Cas12a system is activated, as well as the contrast in oxidase-mimicking capability exhibited by flower-like MnO2 nanozymes when AFM1 is absent or present. When AFM1 is not present in the sample, single-stranded DNA (ssDNA) is degraded by the activated CRISPR-Cas12a, and the solution turns into yellow due to the catalytic activity of the nanozymes. While, in the attendance of AFM1, ssDNA degradation does not occur due to the inactivation of the CRISPR-Cas12a. Therefore, with the adsorption of the ssDNA on the MnO2 nanozymes, their catalytic activity decreases, and the solution color becomes pale yellow due to less oxidation of the chromogenic substrate. In this aptasensor, the relative absorbance changes increased linearly from 6 to 160 ng L-1, and the detection limit was 2.1 ng L-1. The developed aptasensor displays a selective detection performance and a practical application for quantitative analysis of AFM1 in milk samples. The results of the introduced aptasensor open up the way to design other selective and sensitive aptasensors for the detection of other mycotoxins by substitution of the used sequences.

Determination of aflatoxin M1 and ochratoxin A in breast milk in rural centers of Yazd, Iran: Exposure assessment and risk characterization.

Breast milk (BM) is considered as the best source of nutrition which could have prevention effects on various diseases in the first years of a child. Along with nutritive compounds, presence of contaminants such as mycotoxins in BM could be transmitted into neonate. The aim of this study was to determine the occurrence, levels, and factors associated with the presence of aflatoxin M1 (AFM1) and ocratoxin a (OTA) in BM samples of nursing mothers in rural centers of Yazd, Iran. The presence and average AFM1 and OTA concentration in 72 BM samples was measured by competitive ELISA. The demographic and diet parameters of nursing mothers were collected by a questionnaire and were analyzed using SPSS 18 software. AFM1 and OTA were detected in 63 (87.5%) and 47 (65.2%) samples with the mean concentration levels of 19.46 ± 13.26 ng/L (ranges from 5.1 to 53.9) and 200 ± 160 ng/L (ranges from 100 to 2460), respectively. Of these, 32 samples (50.7%) for AFM1 and 23 samples (48.9%) for OTA had values exceeding the limit set by the European Union regulation for infant foods (25 ng/L for AFM1 and 500 ng/L for OTA). It was also found that the risk of AFM1 and OTA occurrence in BM increased significantly with the consumption of beans, bread, cereals, fruit juice and crackers, and cream, respectively. This study showed that the estimated daily intake for AFM1 and OTA by 1 month of age infants was 2.7 and 28.5 ng/kg bw/day, respectively, while, as the age of the infant increased, the values were lower and close to 0.9 and 9.9 ng/kg bw/day for AFM1 and OTA in 12 months of age infants, respectively. The high occurrence and noticeable levels of AFM1 and OTA detected in this study indicated that some infants receive undesirable exposures to AFM1 and OTA with breast milk. Therefore, it is recommended that mothers are advised to avoid certain foods during pregnancy and breastfeeding that are likely sources of mycotoxins.

Density Functional Theory Study on the Interaction between Aflatoxin B1/M1 and Gold Substrate.

Aflatoxin M1 (AFM1) and its precursor, Aflatoxin B1 (AFB1), are highly pathogenic and mutagenic substances, making the detection and sensing of AFB1/M1 a long-standing focus of researchers. Among various detection techniques, surface-enhanced Raman spectroscopy (SERS) is considered an ideal method for AFB1/M1 detection due to its ability not only to enhance characteristic frequencies but also to detect shifts in these frequencies with high repeatability. Therefore, we employed density functional theory in conjunction with surface-enhanced Raman spectroscopy to investigate the interaction between AFB1/M1 and a Au substrate in the context of the SERS effect for the first time. To predict the potential binding sites of AFB1/M1 and Au within the SERS effect, we performed calculations on the molecular electrostatic potential of AFB1/M1. Considering the crucial role of the binding energy in molecular docking studies, we computed the binding energy between two molecules interacting with Au at different binding sites. The molecular frontier orbitals and related chemical parameters of AFB1/M1 and "molecular-Au" complexes were computed to elucidate the alterations in AFB1/M1 molecules under the SERS effect. Subsequently, the theoretical Raman spectra of AFB1/M1 and the complexes were compared and analyzed, enabling determination of the adsorption conformation of AFB1/M1 on the gold surface based on SERS surface selection rules. These findings not only provide a deeper understanding of the interaction mechanism between molecules and substrates in the SERS effect but also offer theoretical support for developing novel aflatoxin SERS sensors.

Chicken CYP1A5 is able to hydroxylate aflatoxin B1 to aflatoxin M1.

Aflatoxin B1 (AFB1), a naturally-occurring mycotoxin, can cause severe toxicological and carcinogenic effects in livestock and humans. Given that the chicken is one of the most important food-producing animals, knowledge regarding AFB1 metabolism and enzymes responsible for AFB1 transformation in the chicken has important implications for chicken production and food safety. Previously, we have successfully expressed chicken CYP1A5 and CYP3A37 monooxygenases in E. coli, and reconstituted them into a functional CYP system consisting of CYP1A5 or CYP3A37, CPR and cytochrome b5. In this study, we aimed to investigate the roles of CYP1A5 and CYP3A37 in the bioconversion of AFB1 to AFM1. Our results showed that chicken CYP1A5 was able to hydroxylate AFB1 to AFM1. The formation of AFM1 followed the typical Michaelis-Menten kinetics. The kinetics parameters of Vmax and Km were determined as 0.83 ± 0.039 nmol/min/nmol P450 and 26.9 ± 4.52 µM respectively. Docking simulations further revealed that AFB1 adopts a "side-on" conformation in chicken CYP1A5, facilitating the hydroxylation of the C9a atom and the production of AFM1. On the other hand, AFB1 assumes a "face-on" conformation in chicken CYP3A37, leading to the displacement of the C9a atom from the heme iron and explaining the lack of AFM1 hydroxylation activity. The results demonstrate that chicken CYP1A5 possesses efficient hydroxylase activity towards AFB1 to form AFM1.

Aflatoxin M1 Analysis in Urine of Mill Workers in Bangladesh: A Pilot Study.

Presence of aflatoxin B1 (AFB1) in food and feed is a serious problem, especially in developing countries. Human exposure to this carcinogenic mycotoxin can occur through dietary intake, but also through inhalation or dermal contact when handling and processing AFB1-contaminated crops. A suitable biomarker of AFB1 exposure by all routes is the occurrence of its hydroxylated metabolite aflatoxin M1 (AFM1) in urine. To assess mycotoxin exposure in mill workers in Bangladesh, we analyzed AFM1 levels in urine samples of this population group who may encounter both dietary and occupational AFB1 exposure. In this pilot study, a total of 76 participants (51 mill workers and 25 controls) were enrolled from the Sylhet region of Bangladesh. Urine samples were collected from people who worked in rice, wheat, maize and spice mills and from controls with no occupational contact to these materials. A questionnaire was used to collect information on basic characteristics and normal food habits of all participants. Levels of AFM1 in the urine samples were determined by a competitive enzyme linked immunosorbent assay. AFM1 was detected in 96.1% of mill workers' urine samples with a range of LOD (40) of 217.7 pg/mL and also in 92% of control subject's urine samples with a range of LOD of 307.0 pg/mL). The mean level of AFM1 in mill workers' urine (106.5 ± 35.0 pg/mL) was slightly lower than that of the control group (123.3 ± 52.4 pg/mL), whilst the mean AFM1 urinary level adjusted for creatinine was higher in mill workers (142.1 ± 126.1 pg/mg crea) than in the control group (98.5 ± 71.2 pg/mg crea). Yet, these differences in biomarker levels were not statistically significant. Slightly different mean urinary AFM1 levels were observed between maize mill, spice mill, rice mill, and wheat mill workers, yet biomarker values are based on a small number of individuals in these subgroups. No significant correlations were found between the study subjects' urine AFM1 levels and their consumption of some staple food items, except for a significant correlation observed between urinary biomarker levels and consumption of groundnuts. In conclusion, this pilot study revealed the frequent presence of AFM1 in the urine of mill workers in Bangladesh and those of concurrent controls with dietary AFB1 exposure only. The absence of a statistical difference in mean biomarker levels for workers and controls suggests that in the specific setting, no extra occupational exposure occurred. Yet, the high prevalence of non-negligible AFM1 levels in the collected urines encourage further studies in Bangladesh regarding aflatoxin exposure.

Assessment of Aflatoxin M1 in human breast and powdered milk in Tehran, Iran.

AIM AND BACKGROUND: Aflatoxins, produced by Aspergillus flavus and Aspergillus parasiticus, are among the most toxic mycotoxins. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1), found in milk and dairy products from animals fed AFB1-contaminated feed. Consumption of AFM1 has related adverse effects on human health. Breast milk can be a source of contamination for infants due to the presence of AFM. AFM1 can also contaminate powdered milk, a significant product of the milk industry. Consequently, monitoring dairy products for these toxins is imperative. STUDY METHOD: A total of 50 samples (25 samples of breast milk and 25 samples of powdered infant milk formula) were collected in Tehran from December 2021 to February 2022. HPLC method was used for the determination of AFM1 in samples. RESULTS: and Discussion: AFM1 was detected in 72% of breast milk samples and 96% of powdered milk samples. AFM1 levels varied significantly between the two sample types (p < 0.05). The average amount of AFM1 in breast milk samples was 25.82 ± 4.72 ng/kg, while the average amount in powdered milk samples was 40.59 ± 7.76 ng/kg. Moreover, 44% of the breast and 68% of powdered milk samples exceeded the AFM1 content limit of the European Union and the Iranian national standard. This study concludes that given the importance of breast milk and formula to maternal and infant health, monitoring and regulating the toxin levels in these products in Tehran is crucial.

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.

Mycotoxin sequestering agent: Impact on health and performance of dairy cows and efficacy in reducing AFM1 residues in milk.

The objectives of this study were to evaluate the exposure to a diet naturally contaminated with mycotoxins on lactation performance, animal health, and the ability to sequester agents (SA) to reduce the human exposure to AFM1. Sixty healthy lactating Holstein cows were randomly assigned to two groups: naturally contaminated diet without and with the addition of a SA (20 g/cow/d AntitoxCooPil® -60% zeolite-40% cell wall-). Each cow was monitored throughout lactation. The concentration of aflatoxin B1 (AFB1) in feed and M1 (AFM1) in milk, health status, and productive and reproductive parameters were measured. AFB1 concentration in feed was very low (2.31 µg/kgDM). The addition of SA reduced the milk AFM1 concentrations (0.016 vs. 0.008 µg/kg) and transfer rates (2.19 vs. 0.77%). No differences were observed in health status, production and reproduction performance. The inclusion of SA in the diet of dairy cows reduce the risk in the most susceptible population.

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.

Regulated and Emerging Mycotoxins in Bulk Raw Milk: What Is the Human Risk?

Mycotoxins are abiotic hazards whose contamination occurs at the pre- and post-harvest stages of the maize value chain, with animal exposure through contaminated feed leading to their excretion into milk. Currently, only aflatoxin M1 is regulated in milk products. Since feed materials and complete feed present a multi-mycotoxin composition and are the main mycotoxin source into milk, it is important to recognize the occurrence of multiple toxins and their co-occurrence in this highly consumed food product. The aim of this study was to determine the content of regulated and emerging mycotoxins in milk samples, which allowed for evaluating the occurrence and co-occurrence patterns of different mycotoxins known to contaminate feed materials and complete animal feed. Human exposure considering the occurrence patterns obtained was also estimated. Aflatoxins, fumonisins, zearalenone, and emerging mycotoxins were among the mycotoxins found to be present in the 100 samples analyzed. Concentrations ranged from 0.006 to 16.3 µg L-1, with no sample exceeding the AFM1 maximum level. Though several mycotoxins were detected, no exceeding values were observed considering the TDI or PMTDI. It can be concluded that the observed exposure does not pose a health risk to milk consumers, though it is important to recognize vulnerable age groups.

Aflatoxin M1 decreases the expression of genes encoding tight junction proteins and influences the intestinal epithelial integrity.

Aflatoxin M1 (AFM1) is a mycotoxin that is commonly found as a milk contaminant, and its presence in milk has been linked to cytotoxicity. The present study aimed to evaluate the acute cytotoxic effects of AFM1 on intestinal Caco-2 cells. Initially, we checked the morphology and viability of Caco-2 cells after treatment with different concentrations of AFM1 (5 ng/L, 50 ng/L, 250 ng/L, 500 ng/L, 1000 ng/L, and 2000 ng/L) for different time intervals (6 h, 12 h, and 24 h). It was found that AFM1 did not show any effect on cell morphology, but 10% decrease in viability above 1000 ng/L after 12 h. Furthermore, DCFDA assay showed increased ROS production after 6 h treatments. qPCR analysis showed an increased expression of epithelial-specific cytoskeleton marker genes, Cytokeratin, Villin, Vimentin, and JAM-1, and a decreased expression of tight junction protein genes, Claudin-1, Occludin, and ZO-1. Similarly, we found an increased expression of Cyp1a1 transcript with an increasing AFM1 concentration and incubation time. This gene expression analysis showed AFM1 can cause disruption of tight junctions between intestinal cells, which was further confirmed by a transwell experiment. In conclusion, consumption of AFM1-contaminated milk does not show any effect on cells morphology and viability but decreases the expression of intestinal barrier transcripts that may lead to the disruption of intestinal barrier function and leaky gut.

Triple strategy-enhanced immunochromatographic assay based on APCB and AIEFM for the ultrasensitive detection of AFM1.

Aflatoxin M1 (AFM1) is highly toxic, widely distributed, and difficult to monitor, posing a serious threat to human health. Therefore, a highly sensitive, rapid, convenient, and low-cost detection method must be urgently established. In this study, a triple strategy-enhanced immunochromatographic assay (ICA) was developed to satisfy these detection requirements. First, a turn-on signal output mode of the fluorescence quenching ICA substituted the turn-off mode of the traditional ICA for sensitive response to trace AFM1, with the limit of detection (LOD) reduced by approximately 4.9-fold. Then, a novel Au and polydopamine (PDA) cogrowth chrysanthemum-like blackbody was prepared as the quenching probe to reduce the background signal. This probe combined the excellent properties of Au nanoparticles with PDA. Thus, its fluorescence quenching constant was higher than that of single Au and PDA nanoparticles by 25.8- and 4.9-fold, respectively. Furthermore, an aggregation-induced emission fluorescence microsphere with a 5.7-fold higher relative quantum yield than a commercial fluorescence microsphere was selected as the signal output carrier to improve the signal-to-noise ratio. The integration of the above triple strategies established a 53.4-fold sensitivity-enhanced fluorescence quenching ICA (LOD = 0.9 pg/mL) for detecting AFM1 in milk, providing a strong technical guarantee for the safety monitoring of milk products.

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.

Detoxification of aflatoxin M1 in different milk types using probiotics.

The aim of this study, research the potential use of probiotics in reducing the toxic effect of Aflatoxin M1 in cow milk, goat milk, sheep milk, and Phosphate-buffered saline (PBS). Milk and Phosphate-buffered saline were contaminated with Aflatoxin M1 at a concentration of 100 ppt. Then, various study groups were formed by adding Lactobacillus acidophilus DSMZ 20079, Lactobacillus rhamnosus GG, and Bifidobacterium bifidum DSMZ 20456 probiotic bacteria at a density of 108 CFU/ml. Then, working groups were stored for 1 day and Aflatoxin M1 levels were analyzed by an Enzyme-Linked Immunosorbent Assay kit. The binding level of Aflatoxin M1 by probiotic bacteria varies between 2.32-12.52% in Phosphate-buffered saline, 9.08-40.14% in cow milk, 15.01-38.01% in goat milk, and 32.49-42.90% in sheep milk. The highest binding level of Aflatoxin M1 was detected in sheep milk and the lowest in Phosphate-buffered saline. The binding ability of Aflatoxin M1 is ranked from highest to lowest in sheep milk, cow milk, and goat milk. The data obtained from this study is important because it is the first study to show that if sheep and goat milk is enriched with probiotics, it can reduce AFM1 exposure.

Novel Porphyrinic Covalent Organic Polymer with Polarity-Switchable Dual Wavelength for Accurate and Sensitive Photoelectrochemical Sensing.

Herein, we synthesized a novel porphyrinic covalent organic polymer (TPAPP-PTCA PCOP) for constructing a polarity-switchable dual-wavelength photoelectrochemical (PEC) biosensor with ferrocene (Fc) and hydrogen peroxide (H2O2) as regulator and amplifier simultaneously. Interestingly, this new PCOP possessed both n-type and p-type semiconductor characteristics, which thus enabled the appearance of a dual-polarity photocurrent at two different excitation wavelengths. Furthermore, Fc and H2O2 could readily switch the photocurrent of PCOP to the cathode and anode stemming from its efficient electron collection and donation function, respectively. Based on these, a PCOP-based PEC biosensor skillfully integrating dual wavelengths with reliable accuracy and polarity switch with high sensitivity was instituted. As a result, the developed PEC biosensor exhibited a low detection limit down to 0.089 pg mL-1 for the most powerful natural carcinogen aflatoxin M1 (AFM1) assay. Impressively, the target exhibited a completely opposite photocurrent difference to the interfering substances, and the linear correlation coefficient of the assay was improved compared to single-wavelength detection. The PEC sensing platform not only provided a basis for exploring multicharacteristic photoactive material but also innovatively developed the detection mode of the PEC biosensor.