Dose and Sequence Dependent Synergism from the Combination of Oxaliplatin with Emetine and Patulin Against Colorectal Cancer.

BACKGROUND: Colorectal cancer is the third most commonly diagnosed cancer in the world, causing many deaths every year. Combined chemotherapy has opened a new horizon in treating colorectal cancer. The objective of the present study is to investigate the activity of oxaliplatin in combination with emetine and patulin against colorectal cancer models. METHODS: IC50 values of oxaliplatin, emetine and patulin were determined against human colorectal cancer cell lines (HT-29 and Caco-2) using MTT reduction assay. Synergistic, antagonistic and additive effects from the selected binary combinations were determined as a factor of sequence of administration and added concentrations. Proteomics was carried out to identify the proteins which were accountable for combined drug action applying to the selected drug combination. RESULTS: Oxaliplatin in combination with patulin produced synergism against human colorectal cancer models depending on dose and sequence of drug administration. Bolus administration of oxaliplatin with patulin proved to be the best in terms of synergistic outcome. Altered expressions of nine proteins (ACTG, PROF1, PPIA, PDIA3, COF1, GSTP1, ALDOA, TBA1C and TBB5) were considered for combined drug actions of oxaliplatin with patulin. CONCLUSION: Bolus administration of oxaliplatin with patulin has the potential to be used in the treatment of colorectal cancer, and would warrant further evaluation using suitable animal model.

Low dose exposure of patulin and protective effect of epicatechin on blood cells in vitro.

In the present study, we aimed to assess antioxidant status in erythrocytes in vitro after patulin (PAT) and epicatechin exposure by measuring antioxidant enzymes (superoxide-dismutase - SOD, glutathione peroxidase - GPx and catalase - CAT) and parameters associated with oxidative stress (malondialdehyde - MDA and ROS). We also investigated the effect of PAT on viability and count of lymphocytes and lymphocyte subpopulations in rabbit blood in vitro. Whole blood of rabbits was used for analysis of antioxidant changes in rabbit erythrocytes after epicatechin and PAT treatment (separately or in combination, at concentrations of 0.2; 2; 20; 200 µg mL-1 of epicatechin and 0.5; 5; 10 µg mL-1 of PAT). Whole blood of rabbits was also used for analysis of count and viability of lymphocytes after PAT treatment at concentrations of 10; 25 and 50 µg mL-1. Results from our experiment confirmed the ability of epicatechin to protect cells against oxidative stress and lipoperoxidation. Our findings indicate that mycotoxin PAT in low concentrations did not affect the activity of antioxidant enzymes in erythrocytes of rabbits significantly. Only slight non-significant changes in lymphocytes count after treatment with low doses of PAT in rabbit blood were observed.

Cytotoxic effects induced by patulin, deoxynivalenol and toxin T2 individually and in combination in hepatic cells (HepG2).

Patulin (PAT), deoxynivalenol (DON) and toxin T-2 (T-2) are mycotoxins distributed worldwide in food and feed. Cytotoxicity of the three mycotoxins individually or in combination in human hepatocellular carcinoma (HepG2) cells was evaluated by MTT assay over 24, 48 and 72 h of exposure. The concentration ranges used were 0.625-15 µM for DON, 1.25-50 nM for T-2 and 0.45-7.5 µM for PAT. The IC50 values obtained ranged from 9.30 to 2.53 µM, from 33.69 to 44.37 nM and from 2.66 to 1.17 µM for DON, T-2 and PAT, respectively. The most cytotoxic mycotoxin to HepG2 cells was T-2 followed by PAT and DON. The combination ratios used for the mixtures were 1:3 (DON: T-2), 1:5 (DON: PAT), 1:1.7 (T-2: PAT) and 1:3:5 (DON: T-2: PAT). The mixture with the highest cytotoxic effect was T-2+PAT, followed by DON + T-2+PAT, DON + T-2 and DON + PAT respect to the cytotoxic effect of their individuals. In the combinations, at low fa an antagonistic effect was detected, and this effect changes the shape of the combination to additive effect at high fa in the mixtures.

Risk assessment of patulin intake through apple-based food by infants and preschool children in Serbia.

This study reports for the first time in Serbia the occurrence of patulin in apple-based food and the risk assessment associated with patulin intake by infants and preschool children. In total, 214 samples of infant fruit juices (48), infant purée (66), and juices for children (small package with straw, 100) were collected over 3 years (2013-15) and analysed using HPLC with ultraviolet detection. Patulin was found in 43.8% of infant juices and 16.7% of infant purée, with all values below the legal limit of 10 µg kg-1 (maximum 8.3 and 7.7 µg kg-1, respectively). The proportion of contaminated samples among fruit juices for children was 43.0%, with the highest patulin concentration at 30.2 µg kg-1, not exceeding the maximum allowed level of 50 µg kg-1. Risk assessment of patulin intake by Serbian infants and preschool children, conducted by deterministic and probabilistic approaches, revealed a hazard quotient well below 1, indicating a tolerable exposure level and no health concern.

Regulation of the antioxidant system in cells of the fission yeast Schizosaccharomyces pombe after combined treatment with patulin and citrinin.

The effects of combined treatment with patulin (PAT) and citrinin (CTN) on Schizosaccharomyces pombe cells were investigated in acute toxicity tests. In comparison with the controls the exposure of fission yeast cells (10(7) cells ml(-1)) to PAT + CTN (250 µM each) for 1 h at a survival rate of 66.6% significantly elevated the concentration of total reactive oxygen species (ROS) via increased levels of peroxides without affecting the concentrations of superoxides or the hydroxyl radical. This treatment induced a 3.08-fold increase in the specific concentration of glutathione and elevated specific activities of catalase and glutathione S-transferase, while at the same time the activity of glutathione reductase decreased. The pattern of the ROS was the same as that induced by CTN (Máté et al., 2014), while the presence of PAT in the PAT + CTN combination treatment modified the activities of the antioxidant system (Papp et al., 2012) in comparison with the individual PAT or CTN treatment, suggesting toxin-specific regulation of glutathione and the enzymes of the antioxidant system and the possibility that the transcription factor (pap1 and atf1) -regulated processes might be influenced directly by ROS.

The potential effect of patulin on mice bearing melanoma cells: an anti-tumour or carcinogenic effect?

Mycotoxins are bioactive compounds that are noxious to human. Their effects on oncogenesis have been satisfactorily elucidated, and some of mycotoxins have been classified as carcinogenic to humans. Nevertheless, patulin (PAT) is considered by the International Agency of Research on Cancer as 'not carcinogenic to humans'. The present study was designed to understand the effect of this mycotoxin on melanoma cells (B16F10) by measuring cell proliferation and assessing the anti-tumour effect in vivo in Balb/c mice. Our results revealed that intraperitoneally administration of PAT for 20 days significantly induces tumour regression in B16F10 cell-implanted mice. This effect was evidenced by the activation of apoptosis which is supported by the increase in p53 and Bax expressions, the downregulation of the protein levels of Bcl2, and the increase in caspase-3 activity. Moreover, systemic toxicity analysis demonstrated that there is no potential toxicity following PAT treatment unlike untreated melanoma mice which suffer from anaemia, inflammation and liver dysfunction. Remarkably, this is the first published report demonstrating the therapeutic efficacy of PAT in vivo models.

Sex-related variations in bone microstructure of rabbits intramuscularly exposed to patulin.

BACKGROUND: Patulin, a toxic mold metabolite, has been found as natural contaminant of processed fruits, most notably apples, apple juices and other apple-based products. A number of adverse health effects in humans and animals are associated with patulin intoxication. The current study was performed to analyse possible toxic effects of patulin on femoral bone microstructure in adult rabbits in detail. Fourteen clinically healthy four-month-old rabbits of both sexes (6 males and 8 females) were included in the study. Animals from the experimental groups (group E♂, n = 3; group E♀, n = 4) were injected intramuscularly with patulin at dose 10 µg/kg body weight two times a week for 28 days. The dose of patulin was estimated based on the maximum permitted level of patulin for apple products intended for infants and young children. Three males and four females without patulin administration served as controls (groups C♂ and C♀). Cortical bone thickness and qualitative and quantitative histological characteristics of compact bone tissue were investigated. RESULTS: Intramuscular applications of patulin significantly increased the thickness of cortical bone in both sexes of rabbits. In patulin-exposed males, an absence of primary vascular longitudinal bone tissue near the endosteal border was observed, which could be associated with intensive bone remodeling. Femoral diaphyses of females displayed a lower number of secondary osteons in the middle part of the substantia compacta, and occurrence of the osteons near the periosteum. This could indicate alterations in bone turnover. Histomorphometrical evaluations showed significantly increased sizes of the primary osteons' vascular canals (P < 0.05) in males exposed to patulin possibly due to mycotoxin-induced increased levels of testosterone. CONCLUSIONS: This study demonstrates significant impact of intramuscular application of patulin on bone microstructure in adult rabbits. Moreover, we have found that the effects of patulin on qualitative and quantitative histological characteristics of compact bone are sex-dependent.

Individual and combined effects of mycotoxins from typical indoor moulds.

The mycotoxins patulin, gliotoxin and sterigmatocystin can be produced by common indoor moulds and enter the human body via inhalation of mycotoxin containing spores and particulates. There are various studies about the individual effects of these mycotoxins, but a lack of knowledge about their effects in mixtures. The aim of this study was to evaluate combined effects on the singe celled organism Tetrahymena pyriformis. Using the MIXTOX model (EU project NOMIRACLE) synergistic or antagonistic effects with dose level deviation or dose ratio dependent deviation were analyzed. The most toxic compound gliotoxin (EC50 0.37 µM) and patulin (EC50 9.3 µM) as shown by the MIXTOX model acted synergistic, caused by similar mode of actions. Within the combination with sterigmatocystin (maximum inhibition of 45% at 12.5 µM) antagonistic effects were observed with switch to synergism if the toxicity of the mixture is mainly caused by sterigmatocystin. In the end the MIXTOX model was applicable for the prediction of combined effects of toxic compounds.

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.

Assessment of dietary intake of patulin from baby foods.

Patulin is a mycotoxin produced by microscopic fungi belonging to the Penicillium and Aspergillus genera, frequently detectable in moldy fruits and their derivatives fruit products. The EC Regulation 1881/06 has imposed the limit for the presence of patulin equal to 10 µg/kg or 10 µg/L in baby food on the basis of a PMTDI of 0.4 µg/kg bw set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). A total of 120 homogenized baby foods were analyzed to evaluate the exposure of baby and children to patulin through the consumption of these products. None of examined samples has shown a toxin concentration above the limit imposed by the law, however a PAT concentration equal to 9 µg/kg was found in 22 samples, slightly below the fixed limit. The presence of patulin in marketed baby food can be regarded as a parameter indicative of the quality of raw materials used.

Effect of patulin on the interdigitating dendritic cells (IDCs) of rat thymus.

Patulin is a common fungal contaminant of ripe apples used for the production of apple juice concentrates and it is also present in other fruits, vegetables and food products. Patulin is a secondary metabolite produced by species of the genera Penicillium, Aspergillus and Byssochlamys. Patulin has been reported to be mutagenic, carcinogenic and teratogenic. Antigen-presenting cells (APCs) are of prime importance in the innate immune response; they capture antigen in tissues and then migrate to the lymphoid organs to present the antigen to T lymphocytes. Thus, they are crucial for the initiation of immunity. Interdigitating dendritic cells (IDCs) are a subset of APCs that are present at the lymphatic organs. In the thymus, they act in positive and negative selection during T cell development. In the present study, patulin was administered orally to growing male rats aged 5-6 weeks. A dose of 0.1 mg kg(-1) bw day(-1) was given to rats for a period of 60 or 90 days daily. The effect of patulin on the IDCs of thymus was investigated by transmission electron microscopy (TEM), and the results were evaluated in terms of cell destruction. In the rats of the control group, it was observed that the IDCs had an indented nucleus, a clear cytoplasm and numerous membrane extensions. In the cytoplasm, a well-developed golgi complex, mitochondria, granular endoplasmic reticulum and a small number of lysosomal structures were observed. At day 60 of patulin-treated rat groups (P-60), loss of cristae in mitochondria and chromatin margination and lysis in the nucleus were found. It was observed that the IDCs had a perinuclear area of cytoplasm surrounded by a peripheral electron-lucent zone. In the cytoplasm of the 90-day patulin-treated rat group (P-90), a peripheral electron-lucent zone was also found, similar to the P-60 group. Additionally increase in vesicular and lysosomal structures, increase in apoptotic bodies and condensation of chromatin in the nucleus were noted. It was observed that patulin leads to apoptotic body formation and cell apoptosis in the IDCs of rat thymus especially in the P-90-treated groups.

Exposure to patulin from consumption of apple-based products.

Patulin is a mycotoxin produced by species of Penicillium, Aspergillus and Byssochylamys. Several Scientific Committees classify patulin as mutagenic, embryotoxic and immunotoxic. It has been found as a natural contaminant of processed apple products and its presence may be indicative of the quality of fruit used in production. In this work, a method for the analysis of patulin is described, based on a simple liquid-liquid extraction with acetonitrile; patulin is analyzed using liquid chromatography with UV detection. Patulin identity was confirmed by GC-MS after its reaction with N-methyl-N-(trimethylsilyl)trifluoroacetamide. Fifty-three apple-containing products were analyzed and patulin was detected in 14 samples in a range 1.5-50.9 microg l(-1); six of which were above the maximum permitted level of the European Union. Based on these results and juice consumption by the Spanish adult population, patulin estimated intake was 0.42 ng kg(-1) body weight per day.

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.

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.

Patulin in domestic and imported apple-based drinks in Belgium: occurrence and exposure assessment.

Apple-based beverages are regularly consumed by adults and children in Belgium. They are locally produced or imported from other countries. The apples used as starting material for these productions are frequently contaminated by mycotoxin-producing moulds and damaged during transport and handling. The current study was undertaken to investigate whether patulin (PAT) is present in the industrial or handicraft-made apple juices and ciders consumed by the Belgian population and to assess the population's exposure to this mycotoxin through apple-based drinks. Belgian (n = 29) and imported (14) apple juices as well as ciders (7) were assayed for PAT by high-performance liquid chromatography with ultraviolet light detection. PAT was detected respectively in 79, 86 and 43% of these tested samples. However, no contaminated sample exceeded the safe level of 50 microg PAT l(-1). Levels of PAT contamination were comparable in Belgian and imported juice samples. The overall mean PAT concentrations were 9.0 and 3.4 microg l(-1) for contaminated apple juices and ciders, respectively. This study also indicates that there was no statistically significant difference in the mean PAT contamination between clear (7.8 microg l(-1)) and cloudy (10.7 microg l(-1)) apple juices, as well as between handicraft-made apple juices (14.6 microg l(-1)) and industrial ones (7.0 microg l(-1)). On the basis of the mean results, a consumer exposure assessment indicates that a daily intake of 0.2 litres apple juice contributes to 45% of the provisional maximum tolerable daily intake for a child of 10 kg body weight.