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PubMed1992

Patulin-induced cellular toxicity: a vital fluorescence study.

The mechanisms of patulin-induced cellular toxicity in an immortalized rat granulosa cell line were examined using several vital fluorescence bioassays. Monochlorobimane and 5-chloromethylfluorescein diacetate were used to monitor cellular glutathione (GSH) levels and revealed dose- and time-dependent depletion of GSH by patulin. A significant reduction in the fluorescence of the monochlorobimane-GSH conjugate by 0.1 microM patulin was observed between 1 and 2 hr. Similar GSH depletion by the mycotoxin was also observed in parallel studies on a liver (Clone 9) and a renal (LLC-PK1) cell line, although reduction of fluorescence occurred within 1 hr at the same dosage. Analysis of the electrical potential-dependent partitioning of rhodamine 123 into mitochondria also revealed significant effects of patulin within 1 hr at 0.1 microM. An initial dose-dependent reduction in mitochondrial fluorescence was followed by loss of selective partitioning of the fluorophore into mitochondria at higher doses and/or a longer exposure of cells to patulin. The reduction in mitochondrial fluorescence was paralleled by a dose-dependent decrease in intracellular pH detected with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Analysis of [Ca2+]i with indo-1 and fluo-3 revealed a significant dose-dependent influx of Ca2+ at 10 microM and an alteration of the pattern of ionomycin-induced Ca2+ influx at 1.0 microM following patulin treatment. A carboxyfluorescein fluorescence photobleaching assay was used to examine the effects of patulin on gap junction-mediated intercellular communication. Dose-dependent reduction in intercellular communication was observed within 2 hr with 1.0 microM patulin. These observations indicate that the fluorescence assays used in this study provide a sensitive index of toxicity caused by exposure to patulin. Further, the toxic effects of patulin may involve direct effects on cellular glutathione levels and mitochondrial function in addition to direct effects on the plasma membrane.

Animals↗
PubMed2004

Investigation of the effects of patulin on thyroid and testis, and hormone levels in growing male rats.

Patulin is a mycotoxin produced by several species of Penicillium, Aspergillus and Byssachlamys. Patulin can be produced on different food products including fruits, grains, cheese, cured meats, but in natural situations patulin is usually found in apple and apple products. In the present study, the time-dependent effects of patulin on the T3, T4, thyroid stimulating hormone, testosterone, luteinizing hormone and growth hormone levels of growing male rats were investigated. Patulin, at a dose of 0.1 mg/kg bw/day, was administered by gavage to growing male rats aged 5-6 weeks for a period of 60 or 90 days. The dose of patulin used in the present study was based on estimated human exposure levels. At the end of the experiment, serum T3, T4, TSH, testosterone, LH and GH levels of rats in control and treatment groups were analysed. In addition, the thyroid and testes were histopathologically examined by light microscopy. Results revealed that while patulin caused an increase (66.6%) in testosterone levels and a decrease (17.3%) in T4 levels of rats treated for 60 days, there was no change in the other hormone levels compared to those of the control group. When patulin treatment was extended to 90 days, increased serum testosterone (75%) and LH levels (146%) were observed. In histological examinations of the testes of rats treated with patulin, oedema, fibrosis and local Leydig cell hyperplasia in the interstitial tissue, and disorganization of seminiferous tubule epithelium were also observed. In addition, the thyroid of rats treated with patulin revealed lymphoid cell inflitration and enlargement of interstitial tissue between follicles, and degenerated colloid.

Animals↗
PubMed2005

Evaluation of several culture media for production of patulin by Penicillium species.

The aim of this study was to evaluate different species of Penicillium to identify those which have the potential to produce the greatest amount of the mycotoxin, patulin. Additionally, six different culture media were compared to determine maximum patulin production. Eleven different strains of Penicillium species were selected because they had previously been reported to be producers of patulin. The strains included Penicillium expansum, Penicillium griseofulvum (formerly Penicillium urticae), Penicillium clavigerum, and Penicillium coprobium and a recent Penicillium sp. isolated from an apple. Cultures were grown in duplicate in three different liquid media: potato dextrose, malt extract, and glucose/yeast extract/peptone, both with and without manganese supplementation. Patulin production was compared at 24, 48, 72, and 96 h. Variability in patulin production occurred among the different species, growth media used, and time of incubation. All three of the P. griseofulvum isolates were the highest producers of patulin at 96 h. For most of the strains, potato dextrose broth supplemented with manganese was optimal for maximum production of patulin. Although P. expansum is frequently cited as the most likely source of patulin in apple juice, certain other Penicillium species are capable of producing more patulin than strains of P. expansum. The apple juice industry should be alert to the possibility that Penicillium species other than P. expansum can be responsible for the occurrence of patulin.

Culture Media↗
PubMed2004

Development of liquid chromatography-electrospray mass spectrometry for the determination of patulin in apple juice: investigation of its contamination levels in Japan.

Patulin is a mycotoxin produced by mainly Penicillium species, for example, P. expansum, and Aspergillus species. There are several reports of patulin contamination in apple juice. Last year, the Ministry of Health, Labour and Welfare of Japan set the maximum allowable level of patulin in apple juice at 50 ppb and decided that the measurement of patulin levels in apple juice products should be conducted. To this end, a simple, accurate, and selective analytical method for the detection of patulin at levels lower than 5 ppb, the detection limit, is desired. This paper reports the development of an analytical method that employs solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS). When MS measurements were conducted with the selected ion monitoring (SIM) mode, the pseudomolecular ions at m/z 153 and 156 were used to monitor patulin and (13)C(3)-labeled patulin, respectively. The detection limit (S/N = 3) and the quantification limit (S/N = 10) of patulin at injection levels into LC-MS were 12.5 and 25 pg, respectively. However, when the actual sample was applied for the analysis based on the developed method including the sample preparation, the detection limit (S/N = 3) and quantification limit (S/N = 10) were 2.5 and 5 pg in sample, respectively. The calibration curve obtained for concentrations ranging from 5 to 500 ppb showed good linearity with a coefficient of determination (r (2)) of 0.999. In addition, the recovery was >95% when an internal standard was used. The method was applied to the analysis of 76 apple juice samples from Japan, and as a result, patulin levels ranging from <1.0 to 45 ppb (detection frequency = 15/76) were detected. In this study, it was found that patulin was a greater contaminant in concentration/reduction than in "not from concentrate" apple juice.

Beverages↗
PubMed2000

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 0.25 g of CCA were continuously loaded with a patulin solution (10 microg/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 microg, 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 microg/ml; CCA adsorption prevented patulin toxicity until 76% breakthrough capacity was achieved. Fixed-bed adsorption with 1.0 g of CCA was also effective in reducing patulin concentrations (20 microg/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.

Adsorption↗
PubMedDate not supplied

Effects of patulin on thymus capillary of rats.

Patulin is a mycotoxin that is produced by species of Penicillum, Aspergillus, and Byssochylamys molds that may grow on a variety of foods including fruit, grains and cheese. Patulin, at a dose of 0.1 mg kg(-1) bw day(-1) was administered orally to growing male rats aged 5-6 weeks for a period of 60 or 90 days. The dose of patulin used in the present study was based on estimated human exposure levels. At the end of these periods, the thymus glands of patulin-treated and control Wistar rats were removed and ultrastructural changes in capillary cells of the thymus of patulin-treated Wistar rats were determined by electron microscopy. The walls of thymus capillaries of the 60-day patulin-treated rat groups (P-60) exhibited degeneration observable in electron microscopic sections. For example, loss of cytoplasm and mitochondrial cristae of cells, swollen endothelial cells, increased thickness of the basement membrane, closed lumen of capillaries, accumulation of fibrous material at the periphery of the capillaries and nuclear anomalies were seen in these sections. Such degeneration and changes were also observed in sections of capillaries of the 90-day patulin-treated rat groups (P-90). The levels of degeneration of endothelial cell nucleus of P-90 were greater than those of P-60. This study demonstrated the ultrastructural degeneration of thymus capillary cells of patulin-treated rats. The results obtained from this study may provide a guide to research dealing with the toxic effects of patulin on tissue and organ ultrastructure.

Administration, Oral↗
PubMed1979

[Determination and the hygienic-toxicologic significance of patulin in fruit and fruit products].

The combination of extracting, chromatographic and fluorescence densitometric steps permits to determine patulin in fruits and fruit products with great precision and sensitivity. The limit of detection is 10 microgram/kg; the recovery rates range from 86 to 92% with a variation coefficient lying between 5.6 and 13.6%. Interferences due to patulin-simulating substances are widely excluded. Patulin concentrations ranging from 0.3 to 42 mg/kg were found in the brown-rotten portions of apples. Juices domestically prepared from healthy fruits and fruits rid of rotten portions, respectively, contained no patulin. On the contrary, patulin concentrations ranging from 0.02 to 0.3 mg/l were observed in commercial apple juices. As to products with subsequent mould infection, patulin values up to 50 mg/l were found in apple juices, and up to 0.4 mg/kg in peach preserves. In a further 24 commercial fruit and vegetable juices, patulin was not detectable even not in cider, in cereals which had gone mouldy spontaneously, and in bread samples. The fruits and fruit products were analysed not only for patulin, but also for aflatoxin and ochratoxin; the latter two, however, were found in none of the samples examined. The possibilities of manufacturing patulin-free products are discussed.

Chromatography, Thin Layer↗
PubMed2005

Patulin reduces glutathione level and enzyme activities in rat liver slices.

In the present study, an attempt was made to identify glutathione (GSH) adducts of patulin in precision-cut rat liver slices, which were used as a model system to study the metabolism and biological effects of this mycotoxin. Patulin disappeared in the slices but none of the GSH adducts, previously demonstrated in the chemical reaction of patulin with GSH, could be detected by HPLC. After incubation with various concentrations of patulin, a concentration-dependent decline of the GSH level was observed in the slices. For example, only 25% of the GSH of controls was found with 200 microM patulin. The activities of glutathione-S-transferase (GST) and of drug metabolizing phase I and phase II enzymes, assayed by the hydroxylation and conjugation of testosterone, were also reduced. On the other hand, incubation with patulin markedly increased lipid peroxidation in the slices. The effects of patulin on enzyme activities and lipid peroxidation may be a consequence of the GSH decline, which cannot be accounted for by a direct reaction of patulin with GSH due to the high concentration of GSH in hepatocytes. The decrease of GSH level and GST activity may be related to the putative mutagenic and carcinogenic potential of patulin.

Animals↗
PubMed2001

Reduction of patulin during apple juice clarification.

Patulin is a mycotoxin produced by a number of molds involved in fruit spoilage. This compound is carcinogenic and teratogenic. Various methods are currently used to reduce the levels of patulin in apple juice, namely, charcoal treatment, chemical preservation (sulfur dioxide), gamma irradiation, fermentation, and trimming of fungus-infected apples. Many of these processes are expensive and time-consuming. Therefore, there is a need to find a convenient and economical process to control patulin levels. This study was undertaken to evaluate the effectiveness of several clarification processes for the reduction of patulin. Clarification was carried out on a laboratory scale. Apple pulp was spiked with patulin, pressed, and clarified using four different processes, namely, fining with bentonite, enzyme (pectinase) treatment, paper filtration, and centrifugation. Patulin was recovered from the clarified juice by liquid-liquid extraction, and solid-phase chromatography was used for sample clean-up prior to analysis by high-performance liquid chromatography (HPLC). The minimum detectable limit using HPLC was 20 microg/liter. Pressing followed by centrifugation resulted in an average toxin reduction of 89%. Total toxin reduction using filtration, enzyme treatment, and fining were 70, 73, and 77%, respectively. Patulin reduction was due to the binding of the toxin to solid substrates that was verified by analyzing the clarified juice as well as the filter cake, pellet, and sediment. The combined concentrations correlated to the spiked concentration. These results reveal that clarification was successful in the reduction of patulin levels in apple juice. However, clarification resulted in high levels of patulin in the pressed pulp after filtration and centrifugation, and this could be harmful if they are used as animal feeds.

Beverages↗
PubMed2000

Micellar electrokinetic capillary electrophoresis for rapid analysis of patulin in apple cider.

A micellar electrokinetic capillary chromatography (MECC) mode was applied to a capillary electrophoresis (CE) method, which was developed for detection and quantitation of patulin in apple ciders. This method used a small sample amount (2 mL) and consumed minimal organic solvent compared to the most commonly used HPLC methods. The sample preparation procedure of the CE method was also simpler than other chromatographic techniques developed for patulin analysis. Patulin was detected with a photodiode array detector at 273 nm. The standard curve was linear (r(2) = 0.9984) from 75 microgram/L to 121 microgram/mL with patulin working solutions corresponding to 3.8 microgram/L to 6.1 microgram/mL patulin in the sample. The linearity was better in a narrower range of concentrations (r(2) = 0.9999) from 75 microgram/L to 24.1 microgram/mL. The limit of detection of the method was 3.8 microgram/L. Patulin recoveries at 4 levels in spiked samples (10-121 microgram/L) ranged from 95.2 to 105.4%. The recoveries were 96. 9% and 99.2% for 2 levels (22.3 and 223 microgram/L, respectively) of patulin in infected apple samples. This method represents a unique alternative method for rapid and sensitive analysis of patulin in apple ciders.

Beverages↗
PubMed2002

Fate of patulin in the presence of the yeast Saccharomyces cerevisiae.

Patulin is known to become analytically non-detectable during the production of cider from contaminated apple juice. The fate of [14C]-labelled patulin during the alcoholic fermentation of apple juice was studied. Three commercial cider strains of Saccharomyces cerevisiae degraded patulin during active fermentative growth, but not when growing aerobically. The products of patulin degradation were more polar than patulin itself and remained in the clarified fermented cider. Patulin did not appear to bind to yeast cells or apple juice sediment in these model experiments. HPLC analysis of patulin-spiked fermentations showed the appearance of two major metabolites, one of which corresponded by both TLC and HPLC to E-ascladiol prepared by the chemical reduction of patulin using sodium borohydride. Using a diode array detector, both metabolites had a lambda(max) = 271 nm, identical to that of ascladiol. The nmr spectrum of a crude preparation of these metabolites showed signals corresponding to those of the E-ascladiol prepared chemically and a weaker set of signals corresponding to those reported in the literature for Z-ascladiol.

Alcoholic Beverages↗
PubMed2002

Co-occurrence of patulin and citrinin in Portuguese apples with rotten spots.

Patulin and citrinin are mycotoxins produced by certain fungi mainly belonging to Penicillium and Aspergillus and may be detected in mouldy fruits and fruit products. The data presented here refer to the simultaneous occurrence of patulin and citrinin in 351 samples of seven different varieties of apples with small rotten areas (Casanova, Golden Delicious, Red Delicious, Reineta, Richared, Rome Beauty, Starking). A rapid multidetection thin layer chromatography (TLC) method was used. The minimum detectable concentrations of patulin and citrinin were 120-130 and 15-20 microg kg(-1) respectively. The percentage contamination with patulin only was higher (68.6%) than that with citrinin only (3.9%). Patulin and citrinin (19.6%) were also detected simultaneously. The highest mean patulin content was 80.50 mg kg(-1) for the Richared variety, but the mean level of citrinin was lower. The lowest mean contaminations of patulin were found in Rome Beauty, Red Delicious and Reineta, ranging from 3.06 to 5.37 mg kg(-1). All analysed apples varieties had low citrinin contamination, ranging from 0.32 to 0.92 mg kg(-1). These findings indicate that there may be a risk of human exposure to patulin through the consumption of juices and jams manufactured with apples with small rotten areas.

Chromatography, Thin Layer↗
PubMed2005

Occurrence of patulin in conventional and organic fruit products in Italy and subsequent exposure assessment.

The occurrence of patulin was investigated in 100 conventional and 69 organic fruity foodstuffs samples commercially available in Italy by using an HPLC method with a limit of quantification of 0.5 microg kg(-1). Patulin was detected in 26 (26%) conventional and 31 (45%) organic products with a significantly higher (p<0.01) mean concentration in the organic products (4.78 vs. 1.15 microg kg(-1)). Mean patulin concentrations in conventional apple juices, pear juices, other juices and fruits purees were 3.14, 0.22, 0.19, 0.11 microg kg(-1), respectively, and 7.11, 11.46, 2.10, 0.18 microg kg(-1) in the relevant organic products. Four samples of juices (one conventional and two organic apple, and one organic pear) contained patulin at concentrations above the limit of 50 microg kg(-1), four at concentrations between 10 and 25 microg kg(-1), and the remaining ones below 10 microg kg(-1). Patulin was detected (<1 microg kg(-1)) in only three of the 23 fruity baby food samples tested (homogenized fruits, 11 conventional and 12 organic). Based on the available data on Italian intakes of fruit juices, the estimated daily intakes of patulin, were 0.38 and 1.57 ng kg(-1) body weight (bw) from conventional and organic products, respectively. Estimated daily intakes of patulin for children were higher, 3.41 ng kg(-1) bw from conventional and 14.17 ng kg(-1) bw from organic products, but largely below the provisional maximum tolerable daily intake (PMTDI) of 400 ng kg(-1) bw. Patulin was also found in two samples of organic apple vinegar (<5 microg kg(-1)) and in fresh apples with rotten spots (12 out of 24 samples) with maximum levels at 16,402 and 44,572 microg kg(-1) for conventional and organic apples, respectively.

Beverages↗
PubMed2001

Incidence of patulin in apple juices marketed in Turkey.

The purpose of this study was to investigate the patulin contamination of apple juices consumed by the Turkish population. Patulin was detected using high-performance liquid chromatography (HPLC) with a UV detector at 280 nm, and the identification of patulin was further confirmed by thin-layer chromatography (TLC). Using HPLC, the recoveries were 79.9 +/- 6.7% and 83.7 +/- 4.6%, and the coefficients of variation were 8.4 and 5.5% for apple juices spiked with the known amounts of patulin (60 and 120 microg/liter. respectively). The minimum patulin level detected was 5 ng in a standard solution and 5 microg/liter in apple juices. The TLC method was used only to confirm patulin levels higher than 20 microg/liter (100 ng/spot) in apple juices. The total number of samples was 45. Patulin was present in detectable levels in 60% of apple juices at concentrations ranging from 19.1 to 732.8 microg/liter. Forty-four percent of the apple juice samples had patulin contamination levels higher than 50 microg/ liter, which is the allowable upper limit in Turkey.

Beverages↗
PubMed2002

Factors affecting patulin production by Penicillium expansum.

Patulin, a mycotoxin produced by Penicillium spp. during fruit spoilage, is a major concern with regard to human health because exposure can result in severe acute and chronic toxicity, including carcinogenic, mutagenic, and teratogenic effects. In this study, we investigated the effects of Penicillium expansum isolate, apple cultivar, storage temperature and time, and pH on the production of patulin. Patulin was analyzed by a previously developed micellar electrokinetic capillary electrophoresis method. P. expansum isolates originating from across Ontario produced widely differing levels of patulin, ranging from 0 to >6 mg/g by dry mycelial weight. The highest patulin levels were those for isolates displaying aggressive growth (characterized by rapidly increasing acidity) accompanied by profuse mycelial development. Distinct patterns in fungal growth rates and patulin production were evident among isolates grown in McIntosh, Empire, and Mutsu ciders. Extensive fungal growth and higher patulin levels (538 to 1,822 microg/ml on day 14) in apple ciders were associated with incubation at room temperature (25 degrees C), although potentially toxic patulin levels (75 to 396 microg/ml on day 24) were also found in refrigerated ciders (4 degrees C) inoculated with P. expansum.

Beverages↗
PubMedDate not supplied

Liquid chromatographic method for determination of patulin in clear and cloudy apple juices and apple puree: collaborative study.

A collaborative trial was conducted to validate the effectiveness of a liquid chromatographic (LC) procedure for determination of patulin in both clear and cloudy apple juices and apple puree. The test portion of clear apple juice was directly extracted with ethyl acetate; cloudy apple juice and apple puree were treated with pectinase enzyme before extraction. After back-extraction into sodium carbonate to remove interfering acidic compounds, the extract was dried and concentrated, and patulin was determined by LC with UV detection. Clear and cloudy apple juices, apple puree test samples naturally contaminated with patulin, and blank test samples for spiking with patulin were sent to 14 collaborators in 12 different European countries. Test portions of each of the 3 test sample types were spiked with patulin at 75 ng/g. Recoveries of patulin ranged from 80 to 92%. Based on the results for spiked test samples (blind pairs) and naturally contaminated test samples (blind pairs at 3 levels), the relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) ranged from 8 to 35% and 11 to 36%, respectively. Although HORRAT values of <1.4 were obtained for all 3 matrixes at patulin levels ranging from 26 to 121 ng/g, better performance values (RSDr values 6-10% and RSDR values 11-25%) were obtained for clear and cloudy apple juice spiked above 50 ng/g, which is either the statutory limit or the advisory level for patulin contamination in apple juices in many countries.

Beverages↗
PubMedDate not supplied

Detoxification of patulin by adduct formation with cysteine.

Patulin adduct was formed by reacting equimolar amounts of patulin and cysteine at pH 6.0. The bactericidal effect of the adduct mixture was less than one hundredth of that of free patulin in plate and liquid cultivation tests with Escherichia coli W 3110 thy pol A1 and pol A1+ strains. The bacterial strains did not liberate free patulin from the adduct mixture present in the growth medium. Neither patulin nor the adduct mixture induced repair effects in E. coli. The oral LD50 of patulin for NMRI male mice was 29 mg/kg. For the adduct mixture, not even as high a dose as 2,370 mg patulin equivalents per kg body weight caused any deaths or macroscopic pathological alteration. Neither patulin nor the adduct mixture was mutagenic to the histidine auxotroph Salmonella typhimurium strains TA 100 and TA 98 in microsomal activation tests or to TA 1950 and 1951 in host-mediated assay.

Animals↗
PubMed1981

Long-term carcinogenicity and toxicity studies of patulin in the rat.

Patulin is a mycotoxin produced by a variety of Penicillium and Aspergillus species which are likely natural contaminants of various foods. The present study was conducted to determine the effects of lifetime administration of patulin in FDRL Wistar rats. Animals received patulin by gastric intubation three times per week at the level of 0.0, 0.1, 0.5 and 1.5 mg per kg body weight. The animals used in this lifetime study were derived from F0 parents exposed to equivalent levels of patulin for 4 weeks before mating, and throughout mating, gestation and lactation. Patulin treatment at 0.5 and 1.5 mg kg-1 to male rats caused a significant decrease in body weight gain in comparison to controls. Body weights of treated female rats were similar to that of control rats. No consistent significant differences among groups were noted in the hematology, clinical chemistry or urine analysis parameters measured during or at the termination of the study. Patulin administered to male and female rats at 1.5 mg kg-1 caused a significantly increased mortality rate as compared to respective control animals. The cause of death appeared to be increased pulmonary and laryngotracheal inflammation. No tumorigenic effect of patulin was observed.

Animals↗