Thermal Stability and Degradation Kinetics of Patulin in Highly Acidic Conditions: Impact of Cysteine.

The thermal stability and degradation kinetics of patulin (PAT, 10 µmol/L) in pH 3.5 of phosphoric-citric acid buffer solutions in the absence and presence of cysteine (CYS, 30 µmol/L) were investigated at temperatures ranging from 90 to 150 °C. The zero-, first-, and second-order models and the Weibull model were used to fit the degradation process of patulin. Both the first-order kinetic model and Weibull model better described the degradation of patulin in the presence of cysteine while it was complexed to simulate them in the absence of cysteine with various models at different temperatures based on the correlation coefficients (R2 > 0.90). At the same reaction time, cysteine and temperature significantly affected the degradation efficiency of patulin in highly acidic conditions (p < 0.01). The rate constants (kT) for patulin degradation with cysteine (0.0036-0.3200 µg/L·min) were far more than those of treatments without cysteine (0.0012-0.1614 µg/L·min), and the activation energy (Ea = 43.89 kJ/mol) was far less than that of treatment without cysteine (61.74 kJ/mol). Increasing temperature could obviously improve the degradation efficiency of patulin, regardless of the presence of cysteine. Thus, both cysteine and high temperature decreased the stability of patulin in highly acidic conditions and improved its degradation efficiency, which could be applied to guide the detoxification of patulin by cysteine in the juice processing industry.

Patulin suppresses α1-adrenergic receptor expression in HEK293 cells.

Patulin (PAT) is a common mycotoxin contaminant of apple products linked to impaired metabolic and kidney function. Adenosine monophosphate activated protein kinase (AMPK), abundantly expressed in the kidney, intercedes metabolic changes and renal injury. The alpha-1-adrenergic receptors (α1-AR) facilitate Epinephrine (Epi)-mediated AMPK activation, linking metabolism and kidney function. Preliminary molecular docking experiments examined potential interactions and AMPK-gamma subunit 3 (PRKAG3). The effect of PAT exposure (0.2-2.5 µM; 24 h) on the AMPK pathway and α1-AR was then investigated in HEK293 human kidney cells. AMPK agonist Epi determined direct effects on the α1-AR, metformin was used as an activator for AMPK, while buthionine sulphoximine (BSO) and N-acetyl cysteine (NAC) assessed GSH inhibition and supplementation respectively. ADRA1A and ADRA1D expression was determined by qPCR. α1-AR, ERK1/2/MAPK and PI3K/Akt protein expression was assessed using western blotting. PAT (1 µM) decreased α1-AR protein and mRNA and altered downstream signalling. This was consistent in cells stimulated with Epi and metformin. BSO potentiated the observed effect on α1-AR while NAC ameliorated these effects. Molecular docking studies performed on Human ADRA1A and PRKAG3 indicated direct interactions with PAT. This study is the first to show PAT modulates the AMPK pathway and α1-AR, supporting a mechanism of kidney injury.

Formation of patulin-glutathione conjugates induced by pulsed light: A tentative strategy for patulin degradation in apple juices.

Patulin is a toxic mycotoxin usually associated with apple products. Due to its unhealthy effects for humans, its content is regulated by the food safety authorities. The removal or degradation of this mycotoxin in contaminated apple juices has been studied with different approaches with uneven effectiveness. However, a strategy based on the chemical reaction between patulin and glutathione (GSH), in order to generate the conjugates that are formed during cell detoxification process, is an innovative approach yet to be evaluated. In this work, the formation of patulin-GSH conjugates activated by the application of pulsed light treatments and catalyzed by Fe2+ ions was evaluated. The study of patulin degradation and effect of the GSH/Fe2+ molar ratio showed that a molar ratio of 5 allows an adequate catalytic effect of the metal ions. In addition, mono-substituted patulin-glutathione adducts were identified as the main type of generated conjugates.

Effective degradation of the mycotoxin patulin in pear juice by porcine pancreatic lipase.

Porcine pancreatic lipase (PPL) was used to degrade the mycotoxin patulin (PAT) in pear juice. The dosage of PPL, the initial concentration of PAT, reaction temperature and time were investigated by batch experiments to study the optimal degradation condition. The concentration of PAT in pear juice was determined by high performance liquid chromatography with an ultraviolet detector (HPLC-UV). The results showed that the optimal condition was 0.02 g PPL/mL pear juice at 40 °C for 24 h. The content of organic acids, volatile flavor components, polyphenols, ascorbic acid and the degree of browning reaction in pear juice, relating to the quality of juice, changed insignificantly. Although the initial PAT concentration was very high, the degradation product was confirmed nontoxic by cytotoxicity test of Caco-2 cells. It suggested that PPL could be further considered to be applied in the degradation of PAT in pear juice.

Identification of Patulin from Penicillium coprobium as a Toxin for Enteric Neurons.

The identification and characterization of fungal commensals of the human gut (the mycobiota) is ongoing, and the effects of their various secondary metabolites on the health and disease of the host is a matter of current research. While the neurons of the central nervous system might be affected indirectly by compounds from gut microorganisms, the largest peripheral neuronal network (the enteric nervous system) is located within the gut and is exposed directly to such metabolites. We analyzed 320 fungal extracts and their effect on the viability of a human neuronal cell line (SH-SY5Y), as well as their effects on the viability and functionality of the most effective compound on primary enteric neurons of murine origin. An extract from P. coprobium was identified to decrease viability with an EC50 of 0.23 ng/µL in SH-SY5Y cells and an EC50 of 1 ng/µL in enteric neurons. Further spectral analysis revealed that the effective compound was patulin, and that this polyketide lactone is not only capable of evoking ROS production in SH-SY5Y cells, but also diverse functional disabilities in primary enteric neurons such as altered calcium signaling. As patulin can be found as a common contaminant on fruit and vegetables and causes intestinal injury, deciphering its specific impact on enteric neurons might help in the elaboration of preventive strategies.

The characteristics, occurrence, and toxicological effects of patulin.

Mycotoxins are the secondary metabolites secreted by different types of fungi to which humans can get exposed mainly via ingestion. Patulin (C7H6O4) is a polyketide lactone produced by various fungal specifies, including Penicillium expansum as the main producer. P. expansum can infect different fruits and vegetables yet it has preference to apples in which they cause blue rot. Therefore, apples and apple-based food products are the main source of Patulin exposure for humans. Patulin was first identified in 1943 under the name of tercinin as a possible antimicrobial agent. Although it is categorized as a non-carcinogen, Patulin has been linked, in the last decades, to neurological, gastrointestinal, and immunological adverse effects, mainly causing liver and kidney damages. In this review, the characteristics of and possible human exposure pathways to Patulin are discussed. Various surveillance and toxicity studies on the levels of Patulin in various food products and effects of Patulin on cells and animal models have been documented as well. Importance of epidemiological studies and a summary of the possible toxicity mechanisms are highlighted with a case study. The commonly used control methods as described in the literature are also discussed to guide future researchers to focus on mitigating mycotoxins contamination in the food industry.

Patulin removal from apple juice using a novel cysteine-functionalized metal-organic framework adsorbent.

Patulin (PAT) is one of the most common toxic contaminants of apple juice, which causes severe food safety issues throughout the apple industry. In order to remove PAT efficiently, a metal-organic framework-based adsorbent (UiO-66(NH2)@Au-Cys) was successfully synthesized and used for PAT removal from juice-pH simulation solution and real apple juice. Batch adsorption experiments were systematically performed to study the adsorption behavior for PAT. The results showed that adsorption process could be well described by the Pseudo-second order model and Freundlich isotherm model. The maximum adsorption capacity (4.38 µg/mg) was 10 times higher than the microbe-based biosorbents. Thermodynamic investigation demonstrated that adsorption process was spontaneous and endothermic. Furthermore, no marked cytotoxicity on NIH 3T3 cell lines was observed when the concentration of the adsorbent was lower than 10 µg/mL. Therefore, UiO-66(NH2)@Au-Cys is a potential adsorbent for PAT removal from apple juice with little quality changes.

Ozone detoxification of patulin in aqueous solution and cytotoxic evaluation using human hepatic carcinoma cells.

Patulin often contaminates fruits and fruit-base products, which seriously threats the health of consumers. In this study, ozone was used to degrade patulin in aqueous solution, and investigated the cytotoxicity of patulin after ozone detoxification on human hepatic carcinoma cells (HepG2) using MTT assay and apoptosis assay. Patulin was rapidly degraded from 24.59 mg/L to 9.85 mg/L within 180 s by 10.60 mg/L of ozone at a flow rate of 90 mL/min, and reduced by 59.94%. The half maximal inhibitory concentration (IC50) of patulin on HepG2 cells was 9.32 µmol/L after 24 h of exposure, and it showed a dose dependent effect. After 90 s of ozone detoxification, the cell viability of HepG2 cells obviously increased from 42.31% to 93.96%, and the total apoptotic cells significantly reduced from 22.24% to 11.18% after 180 s of ozone treatment. The results clearly show the great potential of ozone in degrading patulin in liquid foods.

Effective Adsorption of Patulin from Apple Juice by Using Non-Cytotoxic Heat-Inactivated Cells and Spores of Alicyclobacillus Strains.

Patulin (PAT) is a major threat to many food products, especially apple and apple products, causing human health risks and economic losses. The aim of this study was to remove PAT from apple juice by using the heat-inactivated (HI) cells and spores of seven Alicyclobacillus strains under controlled conditions. The HI cells and spores of seven strains adsorbed PAT effectively, and the HI cells and spores of Alicyclobacillus acidocaldarius DSM 451 (A51) showed maximum PAT adsorption capacity of up to 12.621 µg/g by HI cells and 11.751 µg/g by HI spores at 30 °C and pH 4.0 for 24 h. Moreover, the PAT adsorption process followed the pseudo-first order kinetic model and the Freundlich isotherm model; thermodynamic parameters revealed that PAT adsorption is a spontaneous exothermic physisorption process. The results also indicated that PAT adsorption is strain-specific. The HI cells and spores of Alicyclobacillus strains are non-cytotoxic, and the bioadsorption of PAT did not affect the quality of the juice. Furthermore, the cell wall surface plays an important role in the adsorption process.

Patulin induced ROS-dependent autophagic cell death in Human Hepatoma G2 cells.

Patulin (PAT) is a secondary metabolite produced by certain species of Penicillium, Byssochlamys and Aspergillus. It has been shown to induce liver toxicity, but the possible molecular mechanisms are not completely elucidated. In our study, we treated Human Hepatoma G2 (HepG2) cells by 3-methyladenine (3-MA), an autophagosome formation inhibitor, and rapamycin, an autophagosome formation stimulator. The results showed that 3-MA protected the HepG2 cells against PAT cytotoxicity, while rapamycin decreased the cell viability. Thus, autophagy may play an important role in PAT-induced toxicity. To uncover the mechanism by which cells decrease proliferation and activation of autophagy, we found that collapses of mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level were increased under treatment with PAT. Further, we elucidated that the expression of p-Akt1 and p-MTOR was inhibited during this process. N-acetyl-l-cysteine (NAC), a ROS inhibitor, protected against PAT-induced cytotoxicity, decreased the protein expression of LC3-II, and up-regulated the level of p-Akt1 and p-MTOR. These findings suggested that PAT-induced autophagic cell death was ROS-dependent in HepG2 cells. In conclusion, it is possible that PAT elicited autophagy through ROS-Akt1-MTOR pathway in the HepG2 cells.

Lung Cancer Chemopreventive Activity of Patulin Isolated from Penicillium vulpinum.

Lung cancer is the most lethal form of cancer in the world. Its development often involves an overactivation of the nuclear factor kappa B (NF-κB) pathway, leading to increased cell proliferation, survival, mobility, and a decrease in apoptosis. Therefore, NF-κB inhibitors are actively sought after for both cancer chemoprevention and therapy, and fungi represent an interesting unexplored reservoir for such molecules. The aim of the present work was to find naturally occurring lung cancer chemopreventive compounds by investigating the metabolites of Penicillium vulpinum, a fungus that grows naturally on dung. Penicillium vulpinum was cultivated in Potato Dextrose Broth and extracted with ethyl acetate. Bioassay-guided fractionation of this extract was performed by measuring NF-κB activity using a HEK293 cell line transfected with an NF-κB-driven luciferase reporter gene. The mycotoxin patulin was identified as a nanomolar inhibitor of TNF-α-induced NF-κB activity. Immunocytochemistry and Western blot analyses revealed that its mechanism of action involved an inhibition of p65 nuclear translocation and was independent from the NF-κB inhibitor α (IκBα) degradation process. Enhancing its interest in lung cancer chemoprevention, patulin also exhibited antiproliferative, proapoptotic, and antimigration effects on human lung adenocarcinoma cells through inhibition of the Wnt pathway.

Patulin and ochratoxin A co-occurrence and their bioaccessibility in processed cereal-based foods: A contribution for Portuguese children risk assessment.

Patulin (PAT) and ochratoxin A (OTA) are well known enteropathogenic mycotoxins that are present in several foodstuffs. Processed cereal-based foods are among the first solid foods eaten by children, a particularly vulnerable population group. There is a lack of knowledge related to the co-occurrence of PAT and OTA in food intended for children consumption and their potential interactions during the digestion process. The present study aims to evaluate, for the first time, the co-occurrence of PAT and OTA in processed cereal-based foods for children consumption, the bioaccessibility of these two mycotoxins, and the contribution of the bioaccessibility data for human health risk assessment. PAT and OTA incidence were 75% and 50%, respectively. These mycotoxins co-occurred in 40% of analysed samples. Bioaccessibility assays revealed mean values of 52% and 56% for PAT, alone and combined with OTA; and 100% and 106% for OTA, alone and combined with PAT. Considering the human health risk assessment, and taking into account the co-occurrence and the bioaccessibility results, this study indicates a tolerable exposure to these mycotoxins representing a low risk for Portuguese children. The present work reinforces the importance of a holistic approach for risk assessment which gathers data from occurrence, exposure and bioaccessibility.

The mycotoxin patulin reacts with DNA bases with and without previous conjugation to GSH: implication for related α,ß-unsaturated carbonyl compounds?

The α,ß-unsaturated carbonyl group is recognized as alert for mutagenicity, attributed to (1) its direct reaction with DNA, counteractable by glutathione (GSH), and (2) oxidative stress caused indirectly by GSH depletion. Accordingly, the α,ß,γ,δ-unsaturated lactone patulin (PAT), a mycotoxin detected in fruits and products derived thereof, is known to induce gene, chromosome, and genome mutations in vitro, its mutagenicity correlating inversely with intracellular GSH levels. Thus, the reactivity of PAT against DNA bases and nucleosides in the absence and presence of GSH and glutathione S-transferases (GSTs) was investigated under cell-free conditions using HPLC mass spectrometry techniques for identification of reaction products. Adduct formation with all four nucleobases as well as with purine base nucleosides occurred even in the presence of GSH, revealing several adducts of PAT, mono- and disubstituted with nucleobases/nucleosides as well as novel GSH-PAT adducts. In addition, novel mixed GSH-PAT-nucleobase adducts were observed. These adducts exhibited a ketohexanoic acid-type structure of the PAT molecule, C6 substituted with GSH and linking C1 of PAT with nitrogens of nucleobases/nucleosides via an amide bond. Formation of GSH-PAT-adenine adducts was not prevented by GSTs, and excess of GSH needed to reduce their formation was higher than for PAT-adenine adducts. The formation of mixed GSH-DNA base adducts has not been described for PAT or any other α,ß-unsaturated carbonyl before, although the reaction mechanism seems to be applicable to a variety of α,ß-unsaturated carbonyls occurring in food and in the environment.

Kinetics of patulin degradation in model solution, apple cider and apple juice by ultraviolet radiation.

Patulin is a mycotoxin produced by a wide range of molds involved in fruit spoilage, most commonly by Penicillium expansum and is a health concern for both consumers and manufacturers. The current study evaluated feasibility of monochromatic ultraviolet (UV) radiation at 253.7 nm as a possible commercial application for the reduction of patulin in fresh apple cider and juice. The R-52G MINERALIGHT® UV bench top lamp was used for patulin destruction. It was shown that 56.5%, 87.5%, 94.8% and 98.6% reduction of patulin can be achieved, respectively, in the model solution, apple cider, apple juice without ascorbic acid addition and apple juice with ascorbic acid addition in 2-mm thickness sample initially spiked by 1 mg·L(-1) of patulin after UV exposure for 40 min at UV irradiance of 3.00 mW·cm(-2). A mathematic model to compare the degradation rate and effective UV dose was developed. The effective UV doses that were directly absorbed by patulin for photochemical reaction were 430, 674, 724 and 763 mJ·cm(-3), respectively. The fluence-based decimal reduction time was estimated to 309.3, 31.3, 28.9 and 5.1 mW·cm(-2)·min, respectively, in four media mentioned above. The degradation of patulin followed the first-order reaction model. The time-based and fluence-based reaction rate constants were determined to predict patulin degradation. The time-based reaction rate constant of samples treated in dynamic regime with constant stirring (model solution: 2.95E-4 s(-1), juice: 4.31E-4 s(-1)) were significantly higher than samples treated in static regime (model solution: 2.79E-4 s(-1), juice: 3.49E-4 s(-1), p < 0.05) when applied UV irradiance and sample thickness were consistent. The reaction rate constant of patulin degradation in apple juice was significantly higher than model solution (p < 0.05). Although further investigations are still needed, the results of this study demonstrated that UV radiation may be an effective method for treating patulin-containing apple cider and juice.

DNA damage in organs of mice treated acutely with patulin, a known mycotoxin.

Patulin, a known mycotoxin, is considered a significant contaminant in apples, apple-derived products and feeds. This study investigated the genotoxic effects of patulin in multiple organs (brain, kidney, liver and urinary bladder) of mice using an in vivo comet assay. We assessed the mechanism underlying this genotoxicity by measuring the GSH content and the thiobarbituric acid-reactive species (TBARS) level. Male CF-1 mice were given 1.0-3.75 mg/kg patulin intraperitoneally. The effect of patulin was dose-dependent and the highest patulin dose induced DNA strand breaks in the brain (damage index, DI, in hippocampus increased from 36.2 in control animals to 127.5), liver (44.3-138.4) and kidneys (31.5-99); decreased levels of GSH (hippocampus--from 46.9 to 18.4 nmol/mg protein); and an increase in lipid peroxidation (hippocampus--from 5.8 to 20.3 MDA equivalents/mg protein). This finding establishes an interrelationship between the pro-oxidant and genotoxic effects of patulin. Pre-treatment administration of N-acetyl-cysteine reduced patulin-induced DNA damage (hippocampus--DI from 127.5 to 39.8) and lipid peroxidation (hippocampus--20.3 to 12.8 MDA equivalents/mg protein) by restoring cellular GSH levels, reinforcing the positive relationship between patulin-induced GSH depletion and DNA damage caused by systemic administration of this mycotoxin.

The mycotoxin, patulin, increases the expression of PXR and AhR and their target cytochrome P450s in primary cultured human hepatocytes.

The mycotoxin, patulin (PAT), which is frequently found in apples, grapes, oranges, pear, peaches, and in apple juices, has previously been shown to be cytotoxic, genotoxic, and mutagenic. In this study, we have investigated the effect of PAT on mRNA level of pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and their corresponding target cytochrome P450s. Using primary cultures of adult human hepatocytes, we evaluated PAT cytotoxicity on hepatocytes after 24 hours of treatment. Real time reverse-transcriptase polymerase chain reaction procedure was employed to determine the effect of PAT on receptors (PXR, CAR, and AhR) and cytochrome (CYP3A4, 2B6, 3A5, 2C9, 1A1, and 1A2) genes. Our results showed that PAT reduced hepatocyte viability. At a noncytotoxic range of PAT concentrations, PAT induced an upregulation of the PXR gene in the three treated hepatocytes cultures, whereas CAR was overexpressed in only 1 treated liver. PXR gene induction was accompanied by the enhancement of CYP2B6, 3A5, 2C9, and 3A4 expression. PAT was also found to induce an overexpression of AhR and CYP1A1 and CYP1A2 mRNA expression. These findings suggested that PAT may activate PXR and/or CAR and AhR. However, further investigations are needed to confirm nuclear receptor activation by PAT and to elucidate the molecular mechanism of PAT action.

Identification of an interfering substrate in apple juice and improvement for determination of patulin with high-performance liquid chromatography analyses.

An interfering substance that is not 5-hydroxymethylfurfural appears in some apple juices during high-performance liquid chromatography (HPLC) analysis of patulin based on the AOAC 995.10 method. Because this interfering substance could cause the overestimation of patulin in the apple juices, we tried to identify the substance and to develop an improved method of analyzing patulin free from the influence of this substance. We isolated the substance from the apple juice and identified it as adenosine based on its mass spectrometry, proton nuclear magnetic resonance, and photo diode array spectra. Because of the chemical properties of adenosine, changes in the extraction method under acidic conditions and the HPLC conditions (wavelength and analytical column) were effective for avoiding the influence of adenosine and more specifically for analyzing the patulin. The most effective and simple improvement of the official method was the use of column in-point carbon contents greater than 15.5%.

Analysis of glutathione adducts of patulin by means of liquid chromatography (HPLC) with biochemical detection (BCD) and electrospray ionization tandem mass spectrometry (ESI-MS/MS).

A novel method for the identification of glutathione/electrophile adducts that are inhibiting glutathione-S-transferase (GST) activity was developed and applied for the analysis of the mycotoxin patulin. The method is based on high-performance liquid chromatography (HPLC) coupled to a continuous-flow enzyme reactor serving as biochemical detector (BCD) in parallel to electrospray mass spectrometric detection (ESI-MS). This HPLC-BCD technique combines a separation step and the detection of the inhibition and is therefore ideally suited for the analysis of the activity of single patulin/glutathione adducts within a complex mixture of adducts. Two out of at least 15 detected patulin-glutathione adducts showed strong GST inhibition. In ESI-MS, the inhibitory active adducts were characterized by [M + H]+ ions with m/z 462.1138 and m/z 741.2011, respectively. They could be identified as a dihydropyranone adduct containing one molecule glutathione and a ketohexanoic acid bearing two glutathione molecules.

Mechanism of patulin-induced apoptosis in human leukemia cells (HL-60).

Patulin (PAT) is a fungal secondary metabolite that exhibits potential cellular and animal toxicities. In this study, human promyelocytic leukemia (HL-60) cells were used to elucidate the mechanism and death mode associated with PAT. Morphological evidence of apoptosis, including membrane blebbing, nuclei fragmentation and DNA laddering formation was clearly observed 6h after exposure to PAT. The results of Western blotting indicated that PAT activated various processed caspases, and cleaved DFF45 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner; it also induced a time-dependent increase in caspase 3 and 9 catalytic activities. The apoptosis mediated by PAT in HL-60 was accompanied with cytochrome c release from mitochondria and Bcl-2 expression decrease. The presence of thiol-containing compounds with PAT dramatically reduced the caspase 3 activity that was triggered by PAT; the addition of antioxidants, including mannitol and Tiron, had a similar effect. However, the suppression of p53 protein expression by RNA interference (RNAi) in human embryonic kidney (HEK293) cells did not significantly modify PAT-elicited caspase 3 activity. These findings suggest that PAT-induced apoptosis is mediated through the mitochondrial pathway without the involvement of p53; the interaction with sulfhydryl groups of macromolecules by PAT and the subsequent generation of reactive oxygen species (ROS) plays a primary role in the apoptotic process.

Chemoinformatics analysis identifies cytotoxic compounds susceptible to chemoresistance mediated by glutathione and cystine/glutamate transport system xc-.

Glutathione detoxification has been broadly implicated in resistance to chemotherapy. This study explores the relationship between chemical structure and GSH-mediated chemoresistance. System xc-, the heterodimeric cystine/glutamate exchanger composed of SLC7A11 and SLC3A2, plays a role in maintaining cellular glutathione (GSH) levels. Previous results show that SLC7A11 expression negatively correlates with drug potency across the National Cancer Institute's 60 cell lines for compounds susceptible to GSH-mediated chemoresistance. The number of significant SLC7A11-drug correlations was much greater than those of other genes tested, suggesting that SLC7A11 plays a critical role. Approximately 15% of a curated set of 3045 compounds yielded significant negative SLC7A11 correlations. These compounds tend to contain structural features amenable to GSH reactivity, such as Mannich bases. In cell lines strongly expressing SLC7A11, the potency of selected compounds, was enhanced by inhibition of SLC7A11. This system provides a rapid screen for detecting susceptibility of anticancer drugs to GSH-mediated resistance.