Zinc Supplementation Improves Texture, Oxidative Stability of Caciotta Cheese and Reduces Biogenic Amines Production.

Zinc is essential for animals, playing a vital role in enzyme systems and various biochemical reactions. It is crucial to ensure a sufficient intake of zinc through the diet to maintain efficient homeostasis. Only few studies on zinc effect in cow lactating diet evaluated the effects on milk and cheese quality, with conflicting findings. 24 cows of the Friesian breed were divided into two groups (CTR: control and TRT: treated group). Cows were selected for age, body weight, parity and phase of lactations (mid lactation, 140-160 days). CTR diet contained 38 mg/kg of Zn and TRT diet was supplied with 120 mg/kg of complete feed for 60 days. The objective of current investigation was to evaluate the impact of a dietary Zinc Oxide (ZnO) integration of lactating Friesian cows on chemical composition, zinc content, fatty acid and proteic profile, ammine content, pH, aw, texture, and sensory profile of cheese and to improve the chemical-nutritional quality of milk and cheese. The results showed that ZnO supplementation reduced mesophilic aerobic bacteria and Presumptive Pseudomonas spp. growth, proteolysis, biogenic amines content, lipid oxidation, odour intensity and sour and increased hardness, gumminess, chewiness, elasticity of cheese. Biogenic amines are considered an important aspect of food safety. ZnO integration in cow diet could represent a promising strategy for improving the quality, the safety and shelf-life of caciotta cheese.

Domestic dogs as environmental sentinel in comparative toxicologic pathology: Assessment of metals and rare earth elements concentrations in healthy and neoplastic mammary glands.

Quantification of trace element concentrations in human and animal tissues has acquired great importance in the last few years, considering the pivotal role of these elements in several physiological and pathological processes. Variations in their concentrations appear to have a role in the development and advancement of diseases in both humans and animals, for example, cancer. The purpose of this study was to investigate the concentration of rare earth elements and metals in healthy and neoplastic Formalin-Fixed Paraffin-Embedded (FFPE) mammary gland tissue of dogs. All samples were processed to have a quantitative determination of inorganic elements including metals of known toxicological interest such as Pb, Cd, Tl, As, Hg, the trace elements Mn, Fe, Co, Cu, Zn, Se, and other elements including Cr, V, Mo, Ni, Sb, W, Sn. Moreover, rare earth elements (REEs) (Sc, Y, Lu, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb) were also investigated. Cu and Mo concentrations in mammary cancerous tissue were greater than those in normal mammary glands (p < 0.05). In non-neoplastic tissue increased concentrations of Cd, Co, Ni, Tl, and V were also reported (p < 0.05). The mammary tissue of healthy individuals had greater concentrations of REEs than the neoplastic mammary glands (p < 0.05). The results of our study confirmed differences in mammary inorganic element concentrations between healthy and neoplastic groups, highlighting the potential relevance of these fluctuations in toxicologic pathology.

Comparative analysis of rare earth elements concentrations in domestic dogs and Apennine wolves of Central Italy: Influence of biological, nutritional, and lifestyle factors.

Rare Earth Elements (REEs) are strategical elements playing a crucial role in the industry, especially in producing high-tech materials. Therefore, REEs are new contaminants of emerging concerns. However, due to the lack of exposure data on REE occurrence in environmental matrices, especially in European countries, it is still tricky to establish environmental background levels to assess the ecotoxicological risk related to REEs exposure. The present study aimed to evaluate the liver concentrations of REEs in domestic dogs (Canis lupus familiaris) and Apennine wolves (Canis lupus italicus) living in the Abruzzo region, Italy. Moreover, for the scope of the present study, the dog's group was divided according to their sex, age, lifestyle, and diet. Wolves were categorized concerning their sex and genetic characteristics. Liver samples from dogs and wolves were collected during diagnostic necropsies from carcasses, sample mineralization was performed by a microwave digestion system with a single reaction chamber, and simultaneous determination of the presence of REEs was performed by Inductively Coupled Plasma Mass Spectrometer (Q-ICP-MS) using standard mode for all rare earth elements except scandium (Sc) which was acquired in kinetic energy discrimination (KED) mode. Hepatic concentrations of REEs were statistically significantly higher in wolves compared to dogs. Moreover, significant differences in REEs concentrations arose also from the genetic type of wolf, since "pure wolves" had higher liver concentrations of REEs compared to wolf-dog hybrids. Female and adult dogs also showed elevated REEs compared to male and juvenile dogs, while no significant differences were demonstrated for dogs' diet and lifestyle. The results of the present study confirm the exposure of domestic and wild carnivores to REEs, showing also the ability of REEs to accumulate in carnivore livers, suggesting the potential role of this species as an alternative bioindicator.

Rare earths and other elements content in hen eggs sold in Italy: comparison among main types of farming methods and consumer's health risk assessment.

Recently, in Italy, consumers are choosing hen eggs from farming systems with higher ethical value, due to their perception of a related higher quality and safety. The purposes of this study were to evaluate the existence of differences in elemental content in Italian eggs from organic, barn, and caged hen farming methods and to determine the related potential consumer exposure risk to inorganic contaminants due to the consumption of eggs. One hundred seventy-six egg samples were collected and analyzed using Q-ICP-MS to investigate the content of 14 elements (Pb, As, Hg, Cd, Tl, Fe, Zn, Mn, Cu, Se, Co, Ni, V, and Cr) and 13 rare earth elements (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb). The scenarios of exposure to rare earth and other elements from eggs were estimated for three age groups of consumers. The daily intake values were always lower than the respective safety reference values. In conclusion, Italian hen eggs contain low levels of rare earth and other elements, and therefore, their consumption does not represent a risk of exposure. Finally, no significant differences in contaminants between conventional and organic farming methods were found.

A new method for determining PFASs by UHPLC-HRMS (Q-Orbitrap): Application to PFAS analysis of organic and conventional eggs sold in Italy.

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous environmental pollutants with the ability to uptake to food and feed. Among food, fish, fruits and eggs are considered as major contributors to human dietary exposure. A new method was developed and validated for the simultaneous determination of 18 PFASs in eggs using isotope dilution followed by ultrahigh performance liquid chromatography coupled to high resolution mass spectrometry. The analysis of 132 samples (organic, barn and caged eggs) was performed. Levels were always close to the detection limits and no significant difference emerged among the 3 groups. The highest PFAS concentration in eggs was used to estimate the dietary exposure of different Italian population groups. As expected, children were more highly exposed than adults due to lower body weight. This data suggests that the recent tolerable weekly intake of 4.4 ng kg-1b.w. could be exceeded when the cumulative intake arising from other food products is considered.

Rare Earth Element Variability in Italian Extra Virgin Olive Oils from Abruzzo Region.

Extra virgin olive oil is a food product from the Mediterranean area that is particularly and continuously experiencing to increasing instances of fraudulent geographical labeling. Therefore, origin protection must be improved, mainly based on its intrinsic chemical composition. This study aimed to perform a preliminary chemical characterization of Abruzzo extra virgin olive oils (EVOOs) using rare earth elements (REEs). REEs were evaluated in EVOO samples of different varieties produced in different geographical origins within the Abruzzo region (Italy) in three harvest years using ICP-MS chemometric techniques. Principal component, discriminant, and hierarchical cluster analyses were conducted to verify the influence of the variety, origin, and vintage of the REE composition. The results of a three-year study showed a uniform REE pattern and a strong correlation in most EVOOs, in particular for Y, La, Ce, and Nd. However, europium and erbium were also found in some oil samples. Compared with cultivar and origin, only the harvest year slightly influenced the REE composition, highlighting the interactions of the olive system with the climate and soil chemistry that could affect the multielement composition of EVOOs.

Ion Chromatography-High-Resolution Mass Spectrometry Method for the Determination of Bromide Ions in Cereals and Legumes: New Scenario for Global Food Security.

The new scenario for global food production and supply is decidedly complex given the current forecast of an increase in food fragility due to international tensions. In this period, exports from other parts of the world require different routes and treatments to preserve the food quality and integrity. Fumigation is a procedure used for the killing, removal, or rendering infertile of pests, with serious dangers to human health. The most-used fumigants are methyl bromide and ethylene dibromide. It is important to bear in mind that the soil may contain bromide ions naturally or from anthropogenic source (fertilizers and pesticides that contain bromide or previous fumigations). Different methods (titrimetric, spectrophotometric, and fluorometric approaches) are available to rapidly determine the amount of bromide ion on site in the containers, but these are non-specific and with high limits of quantification. The increasing interest in healthy food, without xenobiotic residues, requires the use of more sensitive, specific, and accurate analytical methods. In order to help give an overview of the bromide ion scenario, a new, fast method was developed and validated according to SANTE 11312/2021. It involves the determination of bromide ion in cereals and legumes through ion chromatography-Q-Orbitrap. The extraction was performed by the QuPPe method, but some modifications were applied based on the matrix. The method described here was validated at four different levels. Recoveries were satisfactory and the mean values ranged between 99 and 106%, with a relative standard deviation lower than 3%. The linearity in the matrix was evaluated to be between 0.010 and 2.5 mg kg-1, with a coefficient of determination (R2) of 0.9962. Finally, the proposed method was applied to different cereals and legumes (rice, wheat, beans, lentils pearled barley, and spelt) and tested with satisfactory results in EUPT-SMR16 organized by EURL.

Brominated and chlorinated contaminants in food (PCDD/Fs, PCBs, PBDD/Fs PBDEs): Simultaneous determination and occurrence in Italian produce.

Validated methodology for the simultaneous determination of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) and polybrominated diphenyl ethers (PBDEs) in foods of animal origin is presented. Method performance indicators were equivalent or better than those required for the control of EU regulated (EU, 2017/644) PCDD/F and PCB congeners in these foods, and for risk assessment through dietary intake. The method uses a high (>90%) proportion of 13Carbon-labelled surrogates for internal standardisation combined with high resolution mass spectrometry that allow accurate quantitation, and this was confirmed by multiple successful participations in proficiency testing for PCDD/Fs, PCBs and PBDEs in food. The same validation and method performance requirements as used for PCDD/Fs were followed for PBDD/Fs. The analysis of a range of food samples (eggs, milk, fish, shellfish, pork, beef and poultry), showed the occurrence of all four classes of contaminants at varying concentration ranges. In general, PCBs were the most prominent contaminant, both, in terms of dioxin-like toxicity, as well as in the occurrence of non-dioxin-like congeners, an observation that concurs with those made in other studies on Italian foods. The levels of PCDD/F and PCB occurrence are consistent with a gradual decline in contamination as reported by some other similar studies. Although all the determined contaminants were detected in the sampled foods, there was poor correlation between the occurrences of the brominated and chlorinated contaminants, and between PBDEs and PBDD/Fs, but better associations were observed between the occurrences of the chlorinated contaminants.

Factors Involved on Tiger-Stripe Foliar Symptom Expression of Esca of Grapevine.

Esca of grapevine causes yield losses correlated with incidence and severity symptom expression. Factors associated with leaf symptom mechanisms are yet to be fully clarified. Therefore, in 2019 and 2020, macro and microelement analyses and leaf reflectance measurements were carried out on leaves at different growth stages in a vineyard located in Abruzzo, central Italy. Surveys were carried out on leaves of both never leaf-symptomatic vines and different categories of diseased vine shoots. Never leaf-symptomatic and diseased vines were also treated with a fertilizer mixture that proved to be able to limit the symptom expression. Results showed that untreated asymptomatic diseased vines had high calcium contents for most of the vegetative season. On the contrary, treated asymptomatic diseased vines showed higher contents of calcium, magnesium, and sodium, at berries pea-sized, before the onset of symptoms. These vines had better physiological efficiency showing higher water index (WI), normalized difference vegetation index (NDVI), and green normalized difference vegetation index (GNDVI) values, compared to untreated asymptomatic vines, at fruit set. Results confirmed the strong response of the plant to symptom expression development and the possibility of limiting this response with calcium and magnesium applications carried out before the symptom onset.

Palytoxin induces dissociation of HSP 27 oligomers through a p38 protein kinase pathway.

Palytoxin (PlTX) induces a stress response in MCF-7 cells that involves the phosphorylation of HSP 27 at serines 15, 78, and 82 by an as yet undetermined mechanism. We have studied the involvement of major groups of the mitogen-activated protein kinase (MAPK) family in this molecular response and focused our analyses on the ERK1/2, JNK, p38 protein kinase (p38K), and ERK5 pathways. The results show that PlTX induces the activation of JNK and p38 kinase but not ERK1/2 and 5 in MCF-7 cells. Through the use of protein kinase inhibitors, we established that blocking p38K, but not JNK, prevents the phosphorylation of HSP 27 induced by PlTX and that MAPKAPK2 participates in the response induced by the toxin under our experimental conditions. The cell death response induced by PlTX was inhibited by preventing JNK phosphorylation but not by blocking p38K/MAPKAPK2 and HSP 27 phosphorylation. Sucrose density gradient centrifugation revealed that MCF-7 cell extracts contain a heterodisperse population of HSP 27, including oligomers and smaller forms. Treating MCF-7 cells with PlTX caused the dissociation of HSP 27 oligomers, and using inhibitors of the JNK and p38K pathways showed that the dissociation of HSP 27 oligomers induced by PlTX involves a p38K-dependent process. We conclude that the changes induced by PlTX in the HSP 27 stress response protein system proceed through a molecular mechanism involving the activation of the p38 kinase pathway and its substrate, MAPKAK2, leading to dissociation of HSP 27 oligomers and the stabilization of a cellular pool of monomers phosphorylated at serines 15, 78 and 82, which could play a protective role against the death response induced by PlTX.

Azaspiracid-1 inhibits the maturation of cathepsin D in mammalian cells.

Azaspiracid-1 (AZA-1) inhibits endocytosis, but the consequences of this alteration on cellular processes are unknown. We hypothesized that the inhibition of endocytosis is a key step of the mode of action of AZA-1, leading to perturbation of cellular processes dependent on proper functioning of endocytic machinery. We tested this working hypothesis by probing whether AZA-1 can alter the maturation of cathepsin D in MCF-7 epithelial cells, as a model system. We found that cell treatment with AZA-1 inhibited the conversion of 52 kDa procathepsin D into the mature 30 kDa protein. The effects induced by AZA-1 were similar to those elicited by chlorpromazine and other agents preventing proper maturation of lysosomal enzymes, indicating that the inhibition of endocytic transfer of proforms to late endosomes/lysosomess is responsible for the effect induced by the toxin. By immunofluorescence microscopy, we found no colocalization of cathepsin D and the early endosomal marker EEA-1 in control cells, where most of cathepsin D resides in late endosomes/lysosomes. Co-localization of cathepsin D and EEA-1 immunoreactivity, in turn, was found in cells exposed to AZA-1, indicating that the toxin blocks protein maturation at the early steps of endocytosis, causing accumulation of procathepsin D in early endosomes. The molecular alteration induced by AZA-1 involved both secreted and intracellular pools of procathepsin D, showing that the toxin effect does not result from a general impairment of vesicular trafficking but is the outcome of a perturbed centripetal process. Furthermore, AZA-1 was found to inhibit procathepsin D maturation also in normal fibroblasts, showing that this molecular response is induced by this toxin in different cell types. The data we obtained corroborated our hypothesis and provide a unified molecular frame for the mode of action of AZAs in animal models, involving a primary alteration of endocytic processes.

Palytoxin induces cell lysis by priming a two-step process in mcf-7 cells.

The cytolytic action of palytoxin (PlTX) was recognized long ago, but its features have remained largely undetermined. We used biochemical, morphological, physiological, and physical tools, to study the cytolytic response in MCF-7 cells, as our model system. Cytolysis represented a stereotyped response induced by the addition of isotonic phosphate buffer (PBS) to cells that had been exposed to PlTX, after toxin removal and under optimal and suboptimal experimental conditions. Cytolysis was sensitive to osmolytes present during cell exposure to PlTX but not in the course of the lytic phase. Fluorescence microscopy showed that PlTX caused cell rounding and rearrangement of the actin cytoskeleton. Atomic force microscopy (AFM) was used to monitor PlTX effects in real time, and we found that morphological and mechanical properties of MCF-7 cells did not change during toxin exposure, but increased cell height and decreased stiffness at its surface were observed when PBS was added to PlTX-treated cells. The presence of an osmolyte during PlTX treatment prevented the detection of changes in morphological and mechanical properties caused by PBS addition to toxin-treated cells, as detected by AFM. By patch-clamp technique, we confirmed that PlTX action involved the transformation of the Na(+),K(+)-ATPase into a channel and found that cell membrane capacitance was not changed by PlTX, indicating that the membrane surface area was not greatly affected in our model system. Overall, our findings show that the cytolytic response triggered by PlTX in MCF-7 cells includes a first phase, which is toxin-dependent and osmolyte-sensitive, priming cells to lytic events taking place in a separate phase, which does not require the presence of the toxin and is osmolyte-insensitive but is accompanied by marked reorganization of actin-based cytoskeleton and altered mechanical properties at the cell's surface. A model of the two-step process of PlTX-induced cytolysis is presented.

The cytolytic and cytotoxic activities of palytoxin.

Palytoxin (PlTX) is one of the largest compound present in nature and, with its strong ability to modify the normal function of different biological systems, is also classified as one of the most potent biotoxins. Many alterations are triggered by PlTX, directly or indirectly related to its interaction with Na(+),K(+)-ATPase and the consequent conversion of this ion pump into a non-specific cation channel. The resulting perturbation of Na(+), K(+), Ca(2+) and H(+) ion fluxes is the driving force of PlTX-induced cytotoxic events, culminating with system disruption and, finally, cell death. The modifications in the distribution of these ions across the plasma membrane play key roles in the promotion of the PlTX-induced cytolytic and cytotoxic responses. In this scenario, PlTX-specific cytolysis can be part, but might not necessarily represent a unique aspect of the cytotoxic effects of the toxin. Owing to the complex array of responses, some of them being cell-type-specific and/or affected by experimental conditions, the distinction between cytolytic and cytotoxic events becomes ill-defined, but the two responses show distinct features, whose further characterization could contribute to a better understanding of the molecular mechanism of cellular effects induced by PlTX.

Azaspiracid-1 inhibits endocytosis of plasma membrane proteins in epithelial cells.

The effect of azaspiracid-1 (AZA-1) on the plasma membrane proteins E-cadherin, Na(+)/K(+)-ATPase, and prolactin receptor (R(prl)) has been investigated in MCF-7 cells. Cell treatment for 24 h with 1nM AZA-1 induced the accumulation of a proteolytic fragment of E-cadherin and significant increases in the levels of Na(+)/K(+)-ATPase and R(prl) at the level of membranous structures. The effect induced by AZA-1 was mimicked by latrunculin A, suggesting that the toxin might act by blocking the endocytosis of plasma membrane proteins. The exposure of intact cells to a biotinylation reagent that does not permeate the plasma membrane provided data showing that AZA-1 treatment of MCF-7 cells doubled the levels of total protein located on the cell surface. The exposure of intact cells to exogenous proteases (trypsin and proteinase K) showed that AZA-1 treatment of MCF-7 cells modifies the availability of the three membrane protein markers to proteolytic attacks, providing evidence that significant portions of the protein pools are located in structures that are not exposed to the cell surface after the treatment with AZA-1. Distinct subcellular locations of the membrane protein markers in MCF-7 cells exposed to AZA-1 were confirmed by immunofluorescence microscopy. Direct evidence that AZA-1 inhibits endocytosis was obtained by showing that AZA-1 blocked the intracellular transfer of E-cadherin-bound antibody in MCF-7 cells. The effects of AZA-1 on the E-cadherin system were confirmed in Caco-2 and Madin Darby canine kidney epithelial cell lines. We conclude that AZA-1 inhibits endocytosis of plasma membrane proteins in epithelial cells.

The cytotoxic pathway triggered by palytoxin involves a change in the cellular pool of stress response proteins.

We have analyzed the proteome of MCF-7 cells exposed to palytoxin (PlTX), to characterize protein components involved in the death response induced by the toxin. The protein profiles of cell lysates were obtained by two-dimensional (2D) electrophoresis, and we found that four components were increased by PlTX treatment. By tryptic digestion of protein spots in the gels and LC-ESI-MS/MS analysis of resulting peptides, those four components were found to include three isoforms of the heat shock protein (hsp) 27 differing with regard to their phosphrylation state, as well as DJ-1/PARK7. The effects exerted by PlTX on hsp 27 and DJ-1 proteins were further quantified by immunoblotting analyses of proteins separated by monodimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and using antibodies recognizing total hsp 27, the hsp 27 forms phosphorylated in Ser(82), and DJ-1 protein. Dose-response and time-course experiments yielded results that only partially confirmed those found by protein staining after 2D electrophoresis. These findings were further checked by immunoblotting of proteins after fractionation by 2D electrophoresis, and we found that only some forms of those comigrating in a single band upon monodimensional SDS-PAGE were actually increased in extracts from PlTX-treated cells. We obtained evidence that the three hsp 27 isoforms whose relative abundance was increased in MCF-7 cells exposed to PlTX comprised two proteins phosphorylated in Ser(82), whereas the third form most likely contains a phosphorylated amino acid residue other than Ser(82). Moreover, we could show that PlTX treatment determined the accumulation of an oxidized isoform of DJ-1 in MCF-7 cells. We conclude that the toxicity pathway of PlTX in MCF-7 cells involves post-translational modifications of hsp 27 and DJ-1 stress response proteins, comprising a shift in the equilibria among hsp 27 isoforms toward those phosphorylated in Ser(82), as well as the oxidation of DJ-1.

A cytolytic assay for the measurement of palytoxin based on a cultured monolayer cell line.

A cytolytic assay that could detect palytoxin and its congeners has been developed by the use of an established cell line grown as monolayer to replace the current hemolytic method. We used MCF-7 cells and cytolysis was measured by the release of cytosolic lactate dehydrogenase (LDH) in the buffer added to treated cells (culture supernatant). A dose-dependent increase in LDH activity in culture supernatants was detected when MCF-7 cells were exposed to palytoxin and its analogue ostreocin D. The cytolytic response induced by palytoxin and ostreocin D was specific for this group of compounds, acting on Na+/K+-ATPase, as it was prevented when cells were preincubated with ouabain. The specificity of our assay for palytoxin and its congeners was confirmed by the finding that cytolysis was not detected when MCF-7 cells were exposed to unrelated toxins such as maitotoxin, tetrodotoxin, okadaic acid, and yessotoxin, even in the case of compounds that elicit cytotoxic responses under our experimental conditions. Using extracts from biological materials after spiking with the palytoxin standard, we found a good correlation between palytoxin levels measured by our cytolytic assay and the expected values. Our cytolytic assay detected palytoxin in naturally contaminated materials, but estimates were significantly higher than the palytoxin contents determined by LC-MS, indicating that naturally contaminated materials contain biologically active palytoxin congeners. We conclude that our cytolytic assay based on the use of MCF-7 cell monolayers is a viable alternative to animal-based methods for the determination of palytoxin and its congeners in contaminated materials.