Probabilistic modeling and risk characterization of the chronic aflatoxin M1 exposure of Hungarian consumers.

Aflatoxin contamination can appear in various points of the food chain. If animals are fed with contaminated feed, AFB1 is transformed-among others-to aflatoxin M1 (AFM1) metabolite. AFM1 is less toxic than AFB1, but it is still genotoxic and carcinogenic and it is present in raw and processed milk and all kinds of milk products. In this article, the chronic exposure estimation and risk characterization of Hungarian consumers are presented, based on the AFM1 contamination of milk and dairy products, and calculated with a probabilistic method, the two-dimensional Monte-Carlo model. The calculations were performed using the R plugin (mc2d package) integrated into the KNIME (Konstanz Information Miner) software. The simulations were performed using data from the 2018-2020 food consumption survey. The AFM1 analytical data were derived from the Hungarian monitoring survey and 1,985 milk samples were analyzed within the framework of the joint project of the University of Debrecen and the National Food Chain Safety Office of Hungary (NÉBIH). Limited AFM1 concentrations were available for processed dairy products; therefore, a database of AFM1 processing factors for sour milk products and various cheeses was produced based on the latest literature data, and consumer exposure was calculated with the milk equivalent of the consumed quantities of these products. For risk characterization, the calculation of hazard index (HI), Margin of Exposure, and the hepatocellular carcinoma incidence were used. The results indicate that the group of toddlers that consume a large amount of milk and milk products are exposed to a certain level of health risk. The mean estimated daily intake of toddlers is in the range of 0.008-0.221 ng kg-1 bw day-1; the 97.5th percentile exposure of toddlers is between 0.013 ng kg-1 bw day-1 and 0.379 ng kg-1 bw day-1, resulting in a HI above 1. According to our study, the exposure of older age groups does not pose an emergent health risk. Nevertheless, the presence of carcinogenic compounds should be kept to a minimum in the whole population.

Physical and Chemical Methods for Reduction in Aflatoxin Content of Feed and Food.

Aflatoxins (AFs) are among the most harmful fungal secondary metabolites imposing serious health risks on both household animals and humans. The more frequent occurrence of aflatoxins in the feed and food chain is clearly foreseeable as a consequence of the extreme weather conditions recorded most recently worldwide. Furthermore, production parameters, such as unadjusted variety use and improper cultural practices, can also increase the incidence of contamination. In current aflatoxin control measures, emphasis is put on prevention including a plethora of pre-harvest methods, introduced to control Aspergillus infestations and to avoid the deleterious effects of aflatoxins on public health. Nevertheless, the continuous evaluation and improvement of post-harvest methods to combat these hazardous secondary metabolites are also required. Already in-use and emerging physical methods, such as pulsed electric fields and other nonthermal treatments as well as interventions with chemical agents such as acids, enzymes, gases, and absorbents in animal husbandry have been demonstrated as effective in reducing mycotoxins in feed and food. Although most of them have no disadvantageous effect either on nutritional properties or food safety, further research is needed to ensure the expected efficacy. Nevertheless, we can envisage the rapid spread of these easy-to-use, cost-effective, and safe post-harvest tools during storage and food processing.

Biological Control and Mitigation of Aflatoxin Contamination in Commodities.

Aflatoxins (AFs) are toxic secondary metabolites produced mostly by Aspergillus species. AF contamination entering the feed and food chain has been a crucial long-term issue for veterinarians, medicals, agroindustry experts, and researchers working in this field. Although different (physical, chemical, and biological) technologies have been developed, tested, and employed to mitigate the detrimental effects of mycotoxins, including AFs, universal methods are still not available to reduce AF levels in feed and food in the last decades. Possible biological control by bacteria, yeasts, and fungi, their excretes, the role of the ruminal degradation, pre-harvest biocontrol by competitive exclusion or biofungicides, and post-harvest technologies and practices based on biological agents currently used to alleviate the toxic effects of AFs are collected in this review. Pre-harvest biocontrol technologies can give us the greatest opportunity to reduce AF production on the spot. Together with post-harvest applications of bacteria or fungal cultures, these technologies can help us strictly reduce AF contamination without synthetic chemicals.

Fat rich diet-induced steatosis and changes in the gut­liver axis / A zsírdús étrend indukálta steatosis és a bél-máj tengely változásai.

INTRODUCTION: Fatty liver can develop as a result of diseases, surgical procedures, medicaments, malnutrition or excessive alcohol consumption, however, fat and poor fiber feeding can be attributed as the primary cause. Non-alcoholic fatty liver can be found in 20-30% of the population. Generally, alimentary-induced fatty liver in early state is described as uncomplicated liver injury. AIM: The aim of our research was to study the effect of fat rich nutrition on the gut-liver axis by routine laboratory, analytical and histological methods in rats. METHODS: We also examined the redox parameters of the liver and of the bowel. Fatty acid composition and element content of liver were measured. RESULTS: Significant changes were found in parameters of redox homeostasis as well as alterations in liver enzymes and metabolites. The changes could be detected in the liver, blood and bowel parts. The development of fatty liver is associated with the decrease of transmethylation capacity. Fatty acid composition and metal ion homeostasis were also altered in liver. Histological examinations showed that hepatocytes were swollen in the central part of the liver lobules, showed droplets and pycnotic nuclei, which were characterized by fatty degeneration. Small and large bowel enterocytes were damaged, sometimes pushed from the surface, and sometimes inflammatory reactions in the mucous membrane were observed. CONCLUSION: Our results suggest that alimentary fatty liver in early state is not considered simply as a reversible alteration because it alters the entire body's redox homeostasis and establishes heart and serious metabolic diseases as well as hasten the development of gastrointestinal tumors. Orv Hetil. 2020; 161(35): 1456-1465.

Patency of uterine wall in pregnancies following assisted and spontaneous conception with antecedent laparoscopic and abdominal myomectomies - a difficult case and systematic review.

A case of uterine rupture at 24 weeks in a pregnancy succeeding myomectomy and triple embryo transfer is described and literature is reviewed systematically to evaluate the importance of uterine rupture in pregnancies after myomectomy in general and some important sub-populations. Systematic search identified 179 papers and following a strategical selection process 45 studies were analyzed in detail, including 6 cohort and 19 observational studies, 3 case series and 17 case reports. Comparison of risk of uterine rupture after abdominal and laparoscopic myomectomy is made. In pregnancies after IVF number of embryos transferred are determined. Optimal contraceptive intervals and surgical techniques are discussed. The consequences of these observations are analyzed and conclusions are made which can assist individualizing treatment options and improve patient selection.

Adverse Effects, Transformation and Channeling of Aflatoxins Into Food Raw Materials in Livestock.

Aflatoxins are wide-spread harmful carcinogenic secondary metabolites produced by Aspergillus species, which cause serious feed and food contaminations and affect farm animals deleteriously with acute or chronic manifestations of mycotoxicoses. On farm, both pre-harvest and post-harvest strategies are applied to minimize the risk of aflatoxin contaminations in feeds. The great economic losses attributable to mycotoxin contaminations have initiated a plethora of research projects to develop new, effective technologies to prevent the highly toxic effects of these secondary metabolites on domestic animals and also to block the carry-over of these mycotoxins to humans through the food chain. Among other areas, this review summarizes the latest findings on the effects of silage production technologies and silage microbiota on aflatoxins, and it also discusses the current applications of probiotic organisms and microbial products in feeding technologies. After ingesting contaminated foodstuffs, aflatoxins are metabolized and biotransformed differently in various animals depending on their inherent and acquired physiological properties. These mycotoxins may cause primary aflatoxicoses with versatile, species-specific adverse effects, which are also dependent on the susceptibility of individual animals within a species, and will be a function of the dose and duration of aflatoxin exposures. The transfer of these undesired compounds from contaminated feed into food of animal origin and the aflatoxin residues present in foods become an additional risk to human health, leading to secondary aflatoxicoses. Considering the biological transformation of aflatoxins in livestock, this review summarizes (i) the metabolism of aflatoxins in different animal species, (ii) the deleterious effects of the mycotoxins and their derivatives on the animals, and (iii) the major risks to animal health in terms of the symptoms and consequences of acute or chronic aflatoxicoses, animal welfare and productivity. Furthermore, we traced the transformation and channeling of Aspergillus-derived mycotoxins into food raw materials, particularly in the case of aflatoxin contaminated milk, which represents the major route of human exposure among animal-derived foods. The early and reliable detection of aflatoxins in feed, forage and primary commodities is an increasingly important issue and, therefore, the newly developed, easy-to-use qualitative and quantitative aflatoxin analytical methods are also summarized in the review.

Niosomes decorated with dual ligands targeting brain endothelial transporters increase cargo penetration across the blood-brain barrier.

Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67 kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles.

From the Cover: In Vitro and In Vivo Blood-Brain Barrier Penetration Studies with the Novel Cyanide Antidote Candidate Dimethyl Trisulfide in Mice.

Recent in vitro and in vivo studies highlight the strong potential of dimethyl trisulfide (DMTS) as an antidote for cyanide (CN) intoxication. Due to its high oxygen demand, the brain is one of the main target organs of CN. The blood-brain barrier (BBB) regulates the uptake of molecules into the brain. In the literature, there is no data about the ability of DMTS to penetrate the BBB. Therefore, our aim was to test the in vitro BBB penetration of DMTS and its in vivo pharmacokinetics in blood and brain. The in vitro BBB penetration of DMTS was measured by using a parallel artificial membrane permeability assay (BBB-PAMPA), and a triple BBB co-culture model. The pharmacokinetics was investigated in a mouse model by following the DMTS concentration in blood and brain at regular time intervals following intramuscular administration. DMTS showed high penetrability in both in vitro systems (apparent permeability coefficients: BBB-PAMPA 11.8 × 10-6 cm/s; cell culture 158 × 10-6 cm/s) without causing cell toxicity and leaving the cellular barrier intact. DMTS immediately absorbed into the blood after the intramuscular injection (5 min), and rapidly penetrated the brain of mice (10 min). In addition to the observed passive diffusion in the in vitro studies, the contribution of facilitated and/or active transport to the measured high permeability of DMTS in the pharmacokinetic studies can be hypothesized. Earlier investigations demonstrating the antidotal efficacy of DMTS against CN together with the present results highlight the promise of DMTS as a brain-protective CN antidote.

Mannich Curcuminoids as Potent Anticancer Agents.

A series of novel curcuminoids were synthesised for the first time via a Mannich-3CR/organocatalysed Claisen-Schmidt condensation sequence. Structure-activity relationship (SAR) studies were performed by applying viability assays and holographic microscopic imaging to these curcumin analogues for anti-proliferative activity against A549 and H1975 lung adenocarcinoma cells. The TNFα-induced NF-κB inhibition and autophagy induction effects correlated strongly with the cytotoxic potential of the analogues. Significant inhibition of tumour growth was observed when the most potent analogue 44 was added in liposomes at one-sixth of the maximally tolerated dose in the A549 xenograft model. The novel spectrum of activity of these Mannich curcuminoids warrants further preclinical investigations.

Effect of solubility enhancement on nasal absorption of meloxicam.

Besides the opioids the standard management of the World Health Organization suggests NSAIDs (non-steroidal anti-inflammatory drugs) alone or in combination to enhance analgesia in malignant and non-malignant pain therapy. The applicability of NSAIDs in a nasal formulation is a new approach in pharmaceutical technology. In order to enhance the nasal absorption of meloxicam (MX) as an NSAID, its salt form, meloxicam potassium monohydrate (MXP), registered by Egis Plc., was investigated in comparison with MX. The physico-chemical properties of the drugs (structural analysis, solubility and dissolution rate) and the mucoadhesivity of nasal formulations were controlled. In vitro and in vivo studies were carried out to determine the nasal applicability of MXP as a drug candidate in pain therapy. It can be concluded that MX and MXP demonstrated the same equilibrium solubility at the pH5.60 of the nasal mucosa (0.017mg/ml); nonetheless, MXP indicated faster dissolution and a higher permeability through the synthetic membrane. The animal studies justified the short Tmax value (15min) and the high AUC of MXP, which is important in acute pain therapy. It can be assumed that the low mucoadhesivity of MXP spray did not increase the residence time in the nasal cavity, and the elimination from the nasal mucosa was therefore faster than in the case of MX. Further experiments are necessary to prove the therapeutic relevance of this MXP-containing innovative intranasal formulation.

A Lipid Base Formulation for Intramuscular Administration of a Novel Sulfur Donor for Cyanide Antagonism.

This study represents a new formulation of the novel Cyanide (CN) antidote, Dimethyl trisulfide (DMTS), for intramuscular administration. This is a naturally occurring organosulfur molecule with the capability of reacting with CN more efficiently than the present sulfur donor type CN therapy of Thiosulfate (TS). Two types of micelles (PEG2000-DSPE and PEG2000-DSPE/TPGS) were prepared and tested for their ability to encapsulate the liquid, highly lipophilic and volatile drug, DMTS. The micellar encapsulation for DMTS does not only eliminate the possible muscle necrosis at the injection sites, but the rate of evaporation within the micelles is suppressed, that can provide a level of stability for the formulation. The method of micelle preparation was optimized and it was demonstrated that the PEG2000-DSPE preparation can dissolve up to 2.0 mg/ml of the antidote candidate. Keeping the injection volume minimized this could provide a maximum DMTS dose of 12.5 mg/kg. However, even this low dose of DMTS showed a remarkable in vivo therapeutic efficacy (2 X LD50 protection) in a mice model when injected intramuscularly. These in vitro and in vivo findings proved the efficacy of DMTS in combating CN intoxication, and the presented work gives valuable insight to micelle preparation and sets the bases for a more advanced future formulation of DMTS.

The Curcumin Analog C-150, Influencing NF-κB, UPR and Akt/Notch Pathways Has Potent Anticancer Activity In Vitro and In Vivo.

C-150 a Mannich-type curcumin derivative, exhibited pronounced cytotoxic effects against eight glioma cell lines at micromolar concentrations. Inhibition of cell proliferation by C-150 was mediated by affecting multiple targets as confirmed at transcription and protein level. C-150 effectively reduced the transcription activation of NFkB, inhibited PKC-alpha which are constitutively over-expressed in glioblastoma. The effects of C-150 on the Akt/ Notch signaling were also demonstrated in a Drosophila tumorigenesis model. C-150 reduced the number of tumors in Drosophila with similar efficacy to mitoxantrone. In an in vivo orthotopic glioma model, C-150 significantly increased the median survival of treated nude rats compared to control animals. The multi-target action of C-150, and its preliminary in vivo efficacy would render this curcumin analogue as a potent clinical candidate against glioblastoma.

ATR-FTIR and Raman spectroscopic investigation of the electroporation-mediated transdermal delivery of a nanocarrier system containing an antitumour drug.

The aim of the present work was the optimization of the transdermal delivery of a lyotropic liquid crystal genistein-based formulation (LLC-GEN). LLC was chosen as medium in view of the poor solubility of GEN in water. Membrane diffusion and penetration studies were carried out with a Franz diffusion cell, through a synthetic membrane in vitro, a chick chorioallantoic membrane ex ovo, and ex vivo excised human epidermis. Thereafter, LLC-GEN was combined with electroporation (EP) to enhance the transdermal drug delivery. The synergistic effect of EP was verified by in vivo ATR-FTIR and ex vivo Raman spectroscopy on hairless mouse skin.

Study of sodium hyaluronate-based intranasal formulations containing micro- or nanosized meloxicam particles.

This article reports on the micro- and nanonization of meloxicam (MEL) with the aim of developing pre-dispersions as intermediates for the design of intranasal formulations. As a new approach, combined wet milling technology was developed in order to reduce the particle size of the MEL. Different milling times resulted in micro- or nanosized MEL in the pre-dispersions with polyvinyl alcohol as stabilizer agent, which were directly used for preparing intranasal liquid formulations with the addition of sodium hyaluronate as mucoadhesive agent. Reduction of the MEL particle size into the nano range led to increased saturation solubility and dissolution velocities, and increased adhesiveness to surfaces as compared with microsized MEL particles. A linear correlation was demonstrated between the specific surface area of MEL and the AUC. The in vitro and in vivo studies indicated that the longer residence time and the uniform distribution of nano MEL spray throughout an artificial membrane and the nasal mucosa resulted in better diffusion and a higher AUC. Nanosized MEL may be suggested for the development of an innovative dosage form with a different dose of the drug, as a possible administration route for pain management.

Ecdysteroids sensitize MDR and non-MDR cancer cell lines to doxorubicin, paclitaxel, and vincristine but tend to protect them from cisplatin.

Ecdysteroids, analogs of the insect molting hormone, are known for their various mild, nonhormonal bioactivities in mammals. Previously, we reported that less-polar ecdysteroids can modulate the doxorubicin resistance of a multidrug resistant (MDR) mouse lymphoma cell line expressing the human ABCB1 transporter. Here, we describe the ability of 20-hydroxyecdysone (1) and its mono- (2) and diacetonide (3) derivatives to sensitize various MDR and non-MDR cancer cell lines towards doxorubicin, paclitaxel, vincristine, or cisplatin. Drug IC50 values with or without ecdysteroid were determined by MTT assay. Compound 3 significantly sensitized all cell lines to each chemotherapeutic except for cisplatin, whose activity was decreased. In order to overcome solubility and stability issues for the future in vivo administration of compound 3, liposomal formulations were developed. By means of their combination index values obtained via checkerboard microplate method, a formulation showed superior activity to that of compound 3 alone. Because ecdysteroids act also on non-ABCB1 expressing (sensitive) cell lines, our results demonstrate that they do not or not exclusively exert their adjuvant anticancer activity as ABCB1 inhibitors, but other mechanisms must be involved, and they opened the way towards their in vivo bioactivity testing against various cancer xenografts.

Sprayed cells and polyelectrolyte films for biomaterial functionalization: the influence of physical PLL-PGA film treatments on dental pulp cell behavior.

Further development of biomaterials is expected as advanced therapeutic products must be compliant to good manufacturing practice regulations. A spraying method for building-up polyelectrolyte films followed by the deposition of dental pulp cells by spraying is presented. Physical treatments of UV irradiation and a drying/wetting process are applied to the system. Structural changes and elasticity modifications of the obtained coatings are revealed by atomic force microscopy and by Raman spectroscopy. This procedure results in thicker, rougher and stiffer film. The initially ordered structure composed of mainly α helices is transformed into random/ß-structures. The treatment enhanced dental pulp cell adhesion and proliferation, suggesting that this system is promising for medical applications.

Sucrose esters increase drug penetration, but do not inhibit p-glycoprotein in caco-2 intestinal epithelial cells.

Sucrose fatty acid esters are increasingly used as excipients in pharmaceutical products, but few data are available on their toxicity profile, mode of action, and efficacy on intestinal epithelial models. Three water-soluble sucrose esters, palmitate (P-1695), myristate (M-1695), laurate (D-1216), and two reference absorption enhancers, Tween 80 and Cremophor RH40, were tested on Caco-2 cells. Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins claudin-1, ZO-1, and ß-catenin. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability for atenolol, fluorescein, vinblastine, and rhodamine 123 in Caco-2 monolayers. No visible opening of the tight junctions was induced by sucrose esters assessed by immunohistochemistry and electron microscopy, but some alterations were seen in the structure of filamentous actin microfilaments. Sucrose esters fluidized the plasma membrane and enhanced the accumulation of efflux transporter ligands rhodamine 123 and calcein AM in epithelial cells, but did not inhibit the P-glycoprotein (P-gp)-mediated calcein AM accumulation in MES-SA/Dx5 cell line. These data indicate that in addition to their dissolution-increasing properties sucrose esters can enhance drug permeability through both the transcellular and paracellular routes without inhibiting P-gp.

Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular vesicles.

Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30-100 nm and 100-1000 nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, renders EV ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3 × 5-5 min global ischemia and reperfusion (IPC) or 30 min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30 min global ischemia and 120 min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9 ± 1.6% vs. 25.0 ± 2.7%), similarly to cardioprotection afforded by IPC (7.3 ± 2.7% vs. 22.1 ± 2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0 ± 2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection.

Pest management systems affect composition but not abundance of phytoseiid mites (Acari: Phytoseiidae) in apple orchards.

We examined the faunal composition and abundance of phytoseiid mites (Acari: Phytoseiidae) in apple orchards under different pest management systems in Hungary. A total of 30 apple orchards were surveyed, including abandoned and organic orchards and orchards where integrated pest management (IPM) or broad spectrum insecticides (conventional pest management) were applied. A total of 18 phytoseiid species were found in the canopy of apple trees. Species richness was greatest in the organic orchards (mean: 3.3 species/400 leaves) and the least in the conventional orchards (1.4), with IPM (2.1) and abandoned (2.7) orchards showing intermediate values. The phytoseiid community's Rényi diversity displayed a similar pattern. However, the total phytoseiid abundance in the orchards with different pest management systems did not differ, with abundance varying between 1.8 and 2.6 phytoseiids/10 leaves. Amblyseius andersoni, Euseius finlandicus, and Typhlodromus pyri were the three most common species. The relative abundance of A. andersoni increased with the pesticide load of the orchards whereas the relative abundance of E. finlandicus decreased. The abundance of T. pyri did not change in the apple orchards under different pest management strategies; regardless of the type of applied treatment, they only displayed greater abundance in five of the orchards. The remaining 15 phytoseiid species only occurred in small numbers, mostly from the abandoned and organic orchards. We identified a negative correlation between the abundance of T. pyri and the other phytoseiids in the abandoned and organic orchards. However, we did not find any similar link between the abundance of A. andersoni and E. finlandicus.