Patulin Toxicity Research Articles

Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p 53 and p 21/WAF1 proteins in skin of mice

Patulin (PAT), a mycotoxin found in apples, grapes, oranges, pear and peaches, is a potent genotoxic compound. WHO has highlighted the need for the study of cutaneous toxicity of PAT as manual labour is employed during pre and post harvest stages, thereby causing direct exposure to skin. In the present study cutaneous toxicity of PAT was evaluated following topical application to Swiss Albino mice. Dermal exposure of PAT, to mice for 4 h resulted in a dose (40–160 μg/animal) and time (up to 6 h) dependent enhancement of ornithine decarboxylase (ODC), a marker enzyme of cell proliferation. The ODC activity was found to be normal after 12 and 24 h treatment of patulin. Topical application of PAT (160 μg/100 μl acetone) for 24–72 h caused (a) DNA damage in skin cells showing significant increase (34–63%) in olive tail moment, a parameter of Comet assay (b) significant G 1 and S-phase arrest along with induction of apoptosis (2.8–10 folds) as shown by annexin V and PI staining assay through flow cytometer. Moreover PAT leads to over expression of p 21/WAF1 (3.6–3.9 fold), pro apoptotic protein Bax (1.3–2.6) and tumor suppressor wild type p 53 (2.8–3.9 fold) protein. It was also shown that PAT induced apoptosis was mediated through mitochondrial intrinsic pathway as revealed through the release of cytochrome C protein in cytosol leading to enhancement of caspase-3 activity in skin cells of mice. These results suggest that PAT has a potential to induce DNA damage leading to p 53 mediated cell cycle arrest along with intrinsic pathway mediated apoptosis that may also be correlated with enhanced polyamine production as evident by induction of ODC activity, which may have dermal toxicological implications.

Mechanisms of Patulin Toxicity under Conditions That Inhibit Yeast Growth

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionMechanisms of Patulin Toxicity under Conditions That Inhibit Yeast GrowthYumiko Iwahashi, Hiroshi Hosoda, Ji-Hyun Park, Joo-Hee Lee, Yoshiteru Suzuki, Emiko Kitagawa, Satomi Mizukami Murata, Nam-Soo Jwa, Man-Bock Gu, and Hitoshi IwahashiCite this: J. Agric. Food Chem. 2007, 55, 4, 1648Publication Date (Web):January 31, 2007Publication History Published online31 January 2007Published inissue 1 February 2007https://doi.org/10.1021/jf063407hCopyright © 2007 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views375Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (20 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Induction of Oxidative Stress Response by the Mycotoxin Patulin in Mammalian Cells

Patulin (PAT), a mycotoxin mainly produced by Penicillium and Aspergillus, is found in various foods and feeds. In the present study, its effects on oxidative stress in various mammalian cell lines were investigated. When cell-permeating fluorescent dyes were used as indicators of the generation of reactive oxygen species (ROS), we found that PAT treatment directly increased intracellular oxidative stress in human embryonic kidney (HEK293) and human promyelocytic leukemia (HL-60) cells. Lipid peroxidation levels were also significantly increased in HL-60 cells and mouse kidney homogenates treated with PAT. Suppression of CuZn-superoxide dismutase (SOD) expression in mammalian cells by small interfering RNA resulted in an increase in PAT-mediated membrane damage, while overexpression of human CuZn-SOD or catalase led to a reduction in damage, indicating the involvement of ROS in PAT toxicity. Pretreatment of HEK293 cells with Tiron, a free radical scavenger, reduced the phosphorylation levels of extracellular signal-regulated kinase (ERK) 1/2 elicited by PAT. The ERK1/2 signaling pathway inhibitor, U0126, also significantly decreased the levels of ROS associated with PAT treatment. These findings indicate that PAT treatment results in the ROS production in mammalian cells, and ROS partially contributes to PAT-induced cytotoxicity. Activation of ERK1/2 signaling pathway is correlated with PAT-mediated ROS.

Evaluation of the reproductive toxicity of patulin in growing male rats

Patulin is a mycotoxin produced by several Penicillium, Aspergillus and Byssachlamys species. Patulin can be produced on different food products including fruits, grains, cheese, cured meats, but in natural situations patulin is exclusively found in apple and apple products. Patulin, at dose of 0.1 mg/kg bw/day, was administered by gavage to the growing male rats aged 5–6 week for 60 or 90 days. At the end of the experiment, sperm counts and morphology were investigated. Also, effects of patulin on the epididymis, seminal vesicle and prostate tissues were examined histopathologically and morphologically. While sperm counts increased in patulin-treated rats for 60 days, sperm counts in patulin-treated rats for 90 days decreased compared to the corresponding control group. Patulin affected sperm morphology of growing male rats. Tail abnormalities like bent and/or coiled tails, and sticking of sperm tails were observed. A significant change was not determined in absolute and relative weights of the seminal vesicle and prostate of patulin-treated rats. While absolute cauda epididymal weights increased in rats treated with patulin for 60 days, absolute and relative cauda epididymal weights reduced in rats treated with patulin for 90 days. In histologic examination, some histopathological changes were observed in the epididymis and prostate tissues of rats in patulin treatment groups.

Mechanisms of Patulin Toxicity under Conditions That Inhibit Yeast Growth

Patulin, 4-hydroxy-4H-furo[3,2c]pyran-2(6H)-one, is one of the best characterized and most widely disseminated mycotoxins found in agricultural products. Nonetheless, the mechanisms by which patulin causes toxicity are not well understood. Thus, the cytotoxicity of patulin was characterized by analysis of the yeast transcriptome upon challenge with patulin. Interestingly, patulin-induced yeast gene expression profiles were found to be similar to gene expression patterns obtained after treatment with the antifungal agricultural chemicals thiuram, maneb, and zineb. Moreover, patulin treatment was found to activate protein degradation, especially proteasome activities, sulfur amino acid metabolism, and the defense system for oxidative stress. Damage to DNA by alkylation was also suggested, and this seemed to be repaired by recombinational and excision repair mechanisms. Furthermore, the results provide potential biomarker genes for the detection of patulin in agricultural products. The results suggest the possibility of applying the yeast transcriptome system for the evaluation of chemicals, especially for natural chemicals that are difficult to get by organic synthesis.

Prevention of patulin toxicity on rumen microbial fermentation by SH-containing reducing agents.

Patulin, a toxic fungal metabolite, negatively affects rumen fermentation. This mycotoxin has also been associated with intoxication cases in cattle. This study investigates the use of SH-containing reducing compounds to prevent patulin's negative effects on the rumen microbial ecosystem. The effect of 50 microg/mL patulin on the fermentation of alfalfa hay was measured in batch cultures with and without reducing agents. Sulfhydryl-containing cysteine and glutathione prevented the negative effects of the toxin on dry matter degradation, gas, and volatile fatty acid production (P < 0.01). However, non-sulfhydryl-containing ascorbic and ferulic acids did not protect against patulin's toxicity (P > 0.01). Patulin was unstable in buffered rumen fluid as the concentration decreased by half after 4 h of incubation. In the presence of sulfhydryl groups, the toxin disappeared rapidly and was not detected after 1 h of incubation. The utilization of sulfhydryl-containing compounds such as cysteine to avert patulin toxicity could have practical implications in ruminant nutrition.

The Mycotoxin Patulin Alters the Barrier Function of the Intestinal Epithelium: Mechanism of Action of the Toxin and Protective Effects of Glutathione

Patulin is a mycotoxin mainly found in apple and apple products. In addition to being toxic for animals, mutagenic, carcinogenic and teratogenic, patulin induces intestinal injuries, including epithelial cell degeneration, inflammation, ulceration, and hemorrhages. In a study of the cellular mechanisms associated with the intestinal toxicity of patulin, two human epithelial intestinal cell lines (HT-29-D4 and Caco-2-14) were exposed to the mycotoxin. Micromolar concentrations of patulin were found to induce a rapid and dramatic decrease of transepithelial resistance (TER) in both cell lines without major signs of toxicity as assessed by the LDH release assay. Since TER reflects the organization of tight junctions, these data indicate that patulin affected the barrier function of the intestinal epithelium. The inhibitory effect of patulin on TER was closely associated with its reactivity for SH groups: (i) cysteine and glutathione prevented the cells from patulin injury; (ii) patulin toxicity was potentiated by buthionine sulfoximine, a specific glutathione-depleting agent; (iii) treatment of the cells with N-ethylmaleimide, a compound known to react with SH groups, resulted in a marked decrease of TER. Moreover, the inhibitory effect of patulin on TER was mimicked and potentiated by phenylarsine oxide, a specific inhibitor of protein tyrosine phosphatase (PTP). This cellular enzyme is a key regulator of intestinal epithelial barrier function. The active site of PTP contains a cysteine residue (Cys215) that is essential for phosphatase activity. Sulfhydryl-reacting compounds such as acetaldehyde decrease TER through covalent modification of Cys215 of PTP. We propose that the toxicity of patulin for intestinal cells involves, among other potential mechanisms, an inactivation of the active site of PTP.

Induction of micronuclei and chromosomal aberrations by the mycotoxin patulin in mammalian cells: role of ascorbic acid as a modulator of patulin clastogenicity.

Patulin is a mycotoxin produced by several species of Penicillium, Aspergillus and BYSSOCHLAMYS: Patulin is a common contaminant of ripe apples used for the production of apple juice concentrates and is also present in other fruits, vegetables and food products. Patulin has been reported to have mutagenic, carcinogenic and teratogenic properties. Nevertheless, these properties are still a matter of debate. In this report, we further investigated the genotoxicity of patulin in mammalian cells by two different approaches. Firstly, we evaluated the induction of micronuclei in cytokinesis-blocked human lymphocytes. This approach is important because available data concerning the genetic toxicity of patulin in human cells is sparse. Secondly, we chose an established model for patulin genotoxicity, i.e. the chromosomal aberration assay in V79 Chinese hamster cells, to clarify whether concomitant exposure to ascorbic acid with the mycotoxin modulates or not the clastogenicity of patulin. The results unequivocally show induction of DNA-damaged cells by patulin as assessed by both cytogenetic assays. In addition, an almost complete abolition of patulin (0.8 microM) clastogenicity was observed in the presence of 80 microM ascorbic acid (P < 0.05), showing that although a genetic risk is present, ascorbic acid could somehow partially modulate this problem.

Development and Characterization of a Carbon-Based Composite Material for Reducing Patulin Levels in Apple Juice

Patulin, a heterocyclic lactone produced by various species of Penicillium and Aspergillus fungi, is often detected in apple juices and ciders. Previous research has shown the effectiveness of granular activated carbon for reducing patulin levels in aqueous solutions, apple juices, and ciders. In this study, ultrafine activated carbon was bonded onto granular quartz to produce a composite carbon adsorbent (CCA) with a high carbonaceous surface area, good bed porosity, and increased bulk density. CCA in fixed-bed adsorption columns was evaluated for efficacy in reducing patulin levels from aqueous solutions and apple juice. Columns containing 1.0, 0.5, and 0.25g of CCA were continuously loaded with a patulin solution (10 μg/ml) and eluted at a flow rate of 1 ml/min. Results indicated that 50% breakthrough capacities for patulin on 1.0-, 0.5-, and 0.25-g CCA columns were 137.5, 38.5, and 19.9 μg, respectively. The effectiveness of CCA to adsorb patulin and prevent toxic effects was confirmed in vitro using adult hydra in culture. Hydra were sensitive to the effects of patulin, with a minimal affective concentration equal to 0.7 μg/ml; CCA adsorption prevented patulin toxicity until 76% breakthrough capacity was achieved. Fixed-bed adsorption with 1.0g of CCA was also effective in reducing patulin concentrations (20 μg/liter) in a naturally contaminated apple juice, and breakthrough capacities were shown to increase with temperature. Additionally, CCA offered a higher initial breakthrough capacity than pelleted activated carbon when compared in parallel experiments. This study suggests that CCA used in fixed-bed adsorption systems effectively reduced patulin levels in both aqueous solutions and naturally contaminated apple juice; however, the appearance and taste of apple juice may be affected by the treatment process.

The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and α-amino groups

As previous studies have indicated a multiple electrophilic reactivity of patulin (PAT) towards simple thiol nucleophiles, we have methodically investigated the ability of PAT to covalently crosslink proteins in vitro. By means of sodium dodecylsulphate polyacrylamide gel electrophoresis, the formation of PAT-induced intermolecular protein-protein crosslinks was clearly demonstrated for bovine serum albumin containing one thiol group per molecule, but also for the thiol-free hen egg lysozyme. Characterization of the crosslink sites was carried out by (1) modulation of the thiol groups with N-ethylimaleimide and 2-iminothiolane; (2) comparison with various known crosslinking agents, i.e. phenylenedimaleimide, glutardialdehyde, and dimethylsuberimidate, and (3) fluorescence incorporation studies using dansyl-labeled amino acids and a fluorescent glutathione derivative. The thiol group of cysteine was preferred for PAT-mediated crosslink reactions, but the side chains of lysine and histidine, and α-amino groups also exhibited reactivity. PAT can act both as a homobifunctional as well as a heterobifunctional crosslinking agent. The initial formation of a monoadduct with a thiol group appears to activate PAT for the subsequent reaction with an amino group, but also leads to rapid loss of further electrophilic properties when no second nucleophile for crosslink completion is available. Studies using microtubule proteins as a protein with experimentally controllable quarternary structure and a proposed cellular target for PAT toxicity emphasized the influence of specific sterical conditions on crosslink formation at low protein concentrations. Non-polymerized microtubule proteins, i.e. tubulin α,β-dimers, formed a defined product with PAT consisting of an intramolecularly crosslinked β-tubulin, whereas guanosine triphosphate- or paclitaxel-induced polymerization to microtubule-like quarternary structures prior to treatment with PAT gave rise to intermolecular crosslink formation between α- and β-tubulin. In contrast, denaturated tubulin yielded none of those two new protein species, but only unspecific intramolecular crosslinks and highly crosslinked aggregates. Thus, in addition to the amino acid composition, the tertiary and quarternary superstructures of proteins appear to markedly influence their reactivity towards PAT. Under appropriate conditions, the generation of protein crosslinks could easily be observed at concentrations of PAT equal to or even below the concentration of the protein. The relevance of these novel reaction pathways of PAT demonstrated in vitro for its in vivo mechanisms of toxicity remains to be investigated.

Elimination of the toxic effects of patulin onBacillus subtilis andB.megaterium by irradiation

The bacteriological toxicity of patulin irradiated in aqueous solutions was investigated using two strains of bacteria, Bacillus subtilis and B.megaterium. The results of the plate cultivation test showed that the growth inhibition of both strains decreased in relation to absorbed dose. A dose of 1.8 kGy was sufficient to eliminate the bacteriological toxicity of patulin at an initial concentration of 125 micrograms/ml. There was good correlation between the concentration of patulin in the irradiated solution as measured spectrophotometrically and that calculated from the diameter of the inhibition zone.

Acute toxicity of patulin and its interaction with penicillic acid in dogs

The acute toxicity of iv-administered patulin and its interaction with ip-injected penicillic acid were investigated in male dogs. The LD 50 of patulin was estimated to be 10·4 mg/kg (95% confidence limits 7·2–15·1 mg/kg). Patulin had severe effects in dogs, particularly on the lungs. Lethargy, anorexia, haematemesis, diarrhoea, pulmonary haemorrhages and oedema, and luminal haemorrhages of the gastro-intestinal tract were common features in dogs given patulin in a dose of 10 mg/kg or more. Sublethal doses (7·5 mg/kg or less) did not have drastic pulmonary effects. Enhancement of patulin toxicity by penicillic acid was indicated by the occurrence of deaths in dogs exposed simultaneously to sublethal doses of both mycotoxins, by the presence in these dogs of toxic signs and lesions resembling those elicited by a single lethal dose of patulin given alone, and by the presence of pulmonary histopathology in dogs receiving both toxins at levels that, given alone, produced no such lesions. Elevated serum alkaline phosphatase in dogs given the mycotoxin combination also suggested possible interaction between the two toxins.

Acute toxicity of patulin in mice and rats*1

Acute toxicity of patulin in mice and rats*1

Acute toxicity of patulin in mice and rats

Patulin had the following 14-day LD 50 values when dissolved in saline (pH 7.2) and given as a single i.p. dose: mice, 7.6 mg/kg and weanling rats, 5.9 mg/kg. Administration by stomach tube increased the single dose LD 50 value to 17 and 108–118 mg/kg in mice and weanling rats, respectively. Neonatal rats given a single dose of patulin 24 h after birth gained less weight (dose-response relationship apparent than controls during the interval from dosing to 21 days of age. The 14-day oral LD 50 in these neonates was 6.8 mg/kg. Patulin potentiated pentobarbital-induced narcosis in adult mice. Because the lethal effect of patulin also was increased by pretreatment with SKF-525A, we suggest that the parent compound, not a metabolite, is the toxic form of this mycotoxin. Death generally occurred within 48–72 h regardless of the route of patulin administration or animal species involved. Focal hepatic necrosis occurred in 10% of the treated mice. Atelectasis was prominent in 64% of the lungs, and alveolar septal congestion and limited intraalveolar hemorrhage were found in an additional 36% of the treated mice and weanling rats. Although stomachs from nurseling rats exposed by gavage to patulin were massively distended with milk, microscopic examination showed no changes other than the extremely stretched wall.

TOXICOLOGICAL STUDY OF PATULIN IN MONKEYS

ABSTRACTPatulin 4‐hydroxy‐4H‐furo [3,2‐c] ‐pyran‐2(6H)‐one, a mycotoxin produced by certain Aspergillus and Penicillium species, has been found as a contaminant of apple products. Patulin has resulted in toxicological effects in mice and rats; however, little information is available on the oral toxicity of patulin to primates. In this study, pig‐tail monkeys (Macaca nemestrina) were given daily oral doses of patulin for a period of 4 wk. Four groups of two monkeys each were used. One group served as a control and the other groups were given daily dosages of either 5, 50 or 500 μg of patulin per kg of body weight. After 4 wk, the monkeys on 500 μg/kg were given daily dosages of 5 mg/kg for an additional two weeks. To administer appropriate dosages, patulin was dissolved in citric acid buffer and applied to sliced freeze‐dried bananas. Blood samples were taken weekly and hematology, serum protein elec‐trophoresis and levels of serum glutamate‐oxalacetate transaminase, alkaline phosphatase, blood urea nitrogen, cholesterol, protein, glucose, sodium and potassium were determined. No statistically significant changes were observed in these physiological parameters, with the exception of AP, which decreased as patulin dosages increased.

Effects of Mycotoxins on the Development of Epiphyllous Buds of Kalanchoe daigremontiana

Summary In Kalanchoe daigremontiana aflatoxin B 1 (100 μg/ml) and rubratoxin (1 μg/ml) inhibit the elongation of roots, patulin (100 μg/ml) reduces particularly strongly the average number of newly formed roots, and rubratoxin B (100 and 10 μg/ml) totally suppresses the development of roots. Citrinin (100 μg/ml) strongly affects the formation of roots. Sterigmatocystin (100 μg/ml) and diacetoxyscirpenol (100 μg/ml) have only moderate effects. It is assumed that the inhibitory action of aflatoxin B 1 , rubratoxin B and diacetoxyscirpenol may be the result of an antagonism to β-indolylacetic acid and that the toxicity of patulin is due to an interaction with sulfhydryl-containing enzymes.

Intermediate-duration toxicity study of patulin in rats.

The toxicity of patulin was studied in two generations of Sprague-Dawley rats over a period of approximately 10 months. Patulin in 1 mM citrate buffer was administered by gavage to FO generation rats at a dose level of 0, 1.5, 7.5, or 15.0 mg/kg a body weight five times a week for 10-14 wk; females were treated seven times a week during pregnancy. High mortality and insufficient progeny in the groups given 7.5 and 15.0 mg/kg made it impossible to continue those two regimens into the second generation. The study was continued for 20-23 wk with F1A generation animals given 1.5 mg/kg and controls. The only lesion found at necropsy that could be attributed to patulin administration was gaseous distention of the gastrointestinal tract, which was probably the result of the antibiotic effect of this mycotoxin on the normal intestinal flora. A decreased weight gain in male rats of the FO generation was dose-related. An impairment in growth rates of F1A and F2A progeny of both sexes was statistically significant at the 1.5 mg/kg dose level. Fetuses taken from patulin-treated females on day 20 of pregnancy were noticeably smaller than controls fetuses and the difference was significant for F2A males. No other teratological abnormalities related to patulin dosing at the 1.5 mg/kg level were observed consistently in either F1A or F2A fetuses. Patulin did not appear to produce dominant lethal effects at dose levels up to 15.0 mg/kg when given by gavage to the males five times a week for 10 or 11 wk.