Novel role for conceptus signals in mRNA expression regulation by DNA methylation in porcine endometrium during early pregnancy†.

During early pregnancy, porcine conceptuses (the embryos with associated membranes) secrete estradiol-17ß (E2)-their major signal for maternal recognition of pregnancy-and prostaglandin E2 (PGE2). Both hormones induce prominent changes of the endometrial transcriptome in vivo. Studies on endometrial pathologies have shown that E2 affects gene expression by epigenetic mechanisms related to DNA methylation. Herein, we determined the effects of E2 and PGE2 alone, and a combined E2 + PGE2 treatment administered into the uterine lumen in vivo on the expression and activity of DNA-methyltransferases (DNMTs) and on CpG methylation patterns of selected genes in porcine endometrium. To compare the effect of treatment with the physiological effect of pregnancy, endometria from day 12 pregnant/cyclic gilts were included. Both E2 and PGE2 significantly reduced the expression of DNMTs. Likewise, the expressions of DNMT1 and DNMT3A were decreased on day 12 of pregnancy compared to the estrous cycle. DNMT activity increased in endometrial samples following E2 treatment and in gilts on day 12 of pregnancy. Treatment with E2 alone and/or simultaneously with PGE2 altered endometrial DNA methylation of CpG sites of ADAMTS20, ADH1C, BGN, PSAT1, and WNT5A. Different CpG methylation patterns of ADAMTS20, BGN, DMBT1, RASSF1, and WNT5A were found in the endometrium on day 12 of pregnancy compared to day 12 of the estrous cycle. Significant correlations were detected between CpG methylation and gene expression for ADAMTS20, ADH1C, BGN, DMBT1, PSAT1, and WNT5A. Our results indicate that CpG methylation induced by embryonic signals may contribute to regulating endometrial gene expression during pregnancy establishment.

Novel insights into conceptus-maternal signaling during pregnancy establishment in pigs.

Pregnancy establishment in mammals, including pigs, requires coordinated communication between developing conceptuses (embryos with associated membranes) and the maternal organism. Porcine conceptuses signalize their presence by secreting multiple factors, of which estradiol-17ß (E2) is considered the major embryonic signal initiating the maternal recognition of pregnancy. During this time, a limited supply of prostaglandin (PGF2α) to the corpora lutea and an increased secretion of luteoprotective factors (e.g., E2 and prostaglandin E2 [PGE2]) lead to the corpus luteum's maintained function of secreting progesterone, which in turn primes the uterus for implantation. Further, embryo implantation is related to establishing an appropriate proinflammatory environment coordinated by the secretion of proinflammatory mediators including cytokines, growth factors, and lipid mediators of both endometrial and conceptus origin. The novel, dual role of PGF2α has been underlined. Recent studies involving high-throughput technologies and sophisticated experimental models identified a number of novel factors and revealed complex relationships between these factors and those already established. Hence, it seems that early pregnancy should be regarded as a sequence of processes orchestrated by pleiotropic factors that are involved in redundancy and compensatory mechanisms that preserve the essential functions critical for implantation and placenta formation. Therefore, establishing the hierarchy between all molecules present at the embryo-maternal interface is now even more challenging.

Selected Fungicides as Potential EDC Estrogenic Micropollutants in the Environment.

An increasing level of pesticide exposition is being observed as a result of the consumption of large amounts of fruits, vegetables and grain products, which are key components of the vegetarian diet. Fungicides have been classified as endocrine-disrupting compounds, but their mechanisms of action have not yet been clarified. The effect of boscalid (B), cyprodinil (C) and iprodione (I) combined with Tamoxifen (T) and 17ß-estradiol (E2) on cell viability, cell proliferation, reporter gene expression, ROS content, the cell membrane's function, cell morphology and antioxidant enzymes gene expression in MCF-7 and T47D-KBluc cell lines were investigated. The cell lines were chosen due to their response to 17ß -estradiol. The selected fungicides are commonly used in Poland to protect crops against fungi. Our results revealed that the studied fungicides caused significant increases in cell viability and proliferation, and estrogenic activity was present in all studied compounds depending on their concentrations. Oxidative stress activated uncontrolled cancer cell proliferation by inducing ROS production and by inhibiting antioxidant defense. Our findings verify that the studied fungicides could possibly exhibit endocrine-disrupting properties and exposure should be avoided.

Pleiotropic role of prokineticin 1 in the porcine endometrium during pregnancy establishment and embryo implantation †.

Acquisition of endometrial receptivity for embryo implantation is one of the crucial processes during pregnancy and is induced mainly by progesterone and enhanced by conceptus signals. Prokineticin 1 (PROK1) is characterized as a secretory protein with diverse functions in various tissues, including the reproductive tract. PROK1, with its receptor PROKR1, are up-regulated in the porcine endometrium during implantation and in women's receptive endometrium and decidua. However, the function of PROK1 in embryo-maternal communication has still not been fully elucidated. Hence, we hypothesize that PROK1 is involved in endometrial receptivity development and implantation in pigs. In this study, using the porcine in vivo model of intrauterine infusions of estradiol-17ß (E2) and prostaglandin E2 (PGE2), we revealed that these hormones elevated endometrial expression of PROK1 and PROKR1 mRNA, respectively. Moreover, E2, acting synergistically with PGE2, increased PROKR1 protein expression. We also evidenced that PROK1-PROKR1 signaling induced expression of following genes and/or proteins CCN2, CDH13, FGF2, NFATC2, ANGPT1, ANGPT2, CDH1, MUC4, SPP1, IFNG, IL6, LIF, LIFR, TNF, TGFB3, and FGF9, as well as phosphorylation of PTK2 and secretion of IL6 and IL11 by endometrial explants in vitro. Ingenuity pathway analysis revealed that functions associated with the PROK1-regulated genes/proteins include cell-to-cell contact, cell attachment, migration and viability, differentiation of epithelial tissue, leukocyte migration, inflammatory response, angiogenesis, and vasculogenesis. Summarizing, our study suggests that PROK1 acts pleiotropically as an embryonic signal mediator that regulates endometrial receptivity by increasing the expression of the genes and proteins involved in implantation and pregnancy establishment in pigs.

Synergistic action of estradiol and PGE2 on endometrial transcriptome in vivo resembles pregnancy effects better than estradiol alone†.

Successful pregnancy establishment in mammals depends on numerous interactions between embryos and the maternal organism. Estradiol-17ß (E2) is the primary embryonic signal in the pig, and its importance has been questioned recently. However, E2 is not the only molecule of embryonic origin. In pigs, prostaglandin E2 (PGE2) is abundantly synthesized and secreted by conceptuses and endometrium. The present study aimed to determine the role of PGE2 and its simultaneous action with E2 in changes in porcine endometrial transcriptome during pregnancy establishment. The effects of PGE2 and PGE2 acting with E2 were studied using an in vivo model of intrauterine hormone infusions, and were compared to the effects of E2 alone and conceptuses' presence on day 12 of pregnancy. The endometrial transcriptome was profiled using gene expression microarrays followed by statistical analyses. Downstream analyses were performed using bioinformatics tools. Differential expression of selected genes was verified by quantitative polymerase chain reaction. Microarray analysis revealed 2413 differentially expressed genes (DEGs) in the endometrium treated simultaneously with PGE2 and E2 (P < 0.01). No significant effect of PGE2 administered alone on endometrial transcriptome was detected. Gene ontology annotations enriched for DEGs were related to multiple processes such as: focal adhesion, vascularization, cell migration and proliferation, glucose metabolism, tissue remodeling, and activation of immune response. Simultaneous administration of E2 and PGE2 induced more changes within endometrial transcriptome characteristic to pregnancy than infusion of E2 alone. The present findings suggest that synergistic action of estradiol-17ß and PGE2 resembles the effects of pregnancy on endometrial transcriptome better than E2 alone.

Estradiol-17ß Regulates Expression of Luteal DNA Methyltransferases and Genes Involved in the Porcine Corpus Luteum Function In Vivo.

The corpus luteum (CL) is a temporary endocrine gland vital for pregnancy establishment and maintenance. Estradiol-17ß (E2) is the major embryonic signal in pigs supporting the CL's function. The mechanisms of the luteoprotective action of E2 are still unclear. The present study aimed to determine the effect of E2 on luteal expression of factors involved in CL function. An in vivo model of intrauterine E2 infusions was applied. Gilts on day 12 of pregnancy and the estrous cycle were used as referential groups. Concentrations of E2 and progesterone were elevated in CLs of gilts receiving E2 infusions, compared to placebo-treated gilts. Estradiol-17ß stimulated luteal expression of DNA-methyltransferase 1 (DNMT1), but decreased expression of DNMT3B gene and protein, as well as DNMT3A protein. Similar results for DNMT3A and 3B were observed in CLs on day 12 of pregnancy compared to day 12 of the estrous cycle. Intrauterine infusions of E2 altered luteal expression of the genes involved in CL function: PTGFR, PTGES, STAR, HSD17B1, CYP19A1, and PGRMC1. Our findings indicate a role for E2 in expression regulation of factors related to CL function and a novel potential for E2 to regulate DNA methylation as putative physiological mechanisms controlling luteal gene expression.

Prokineticin 1-prokineticin receptor 1 signaling promotes angiogenesis in the porcine endometrium during pregnancy†.

Pregnancy establishment in mammals, including pigs, requires proper communication between embryos and the maternal reproductive tract. Prokineticin 1 (PROK1) has been described as a secretory protein with pleiotropic functions and as a novel tissue-specific angiogenic factor. However, despite the studies performed mainly on human cell lines and in mice, the function of PROK1 in the endometrium during early pregnancy is still not fully elucidated. We hypothesized that PROK1 contributes to pregnancy establishment in pigs. The present study is the first to report that the expression of PROK1 and its receptor (PROKR1) is elevated in the porcine endometrium during the implantation and early placentation period. PROK1 protein was detected mainly in luminal epithelial cells, glandular epithelial cells, and blood vessels in the endometrium. Using the porcine in vivo model of unilateral pregnancy, we revealed that conceptuses induced the endometrial expression of PROK1 and PROKR1. Moreover, the embryonic signal, estradiol-17ß, as well as progesterone, stimulated the endometrial expression of PROK1 and PROKR1. We also evidenced that PROK1-PROKR1 signaling supports endometrial angiogenesis in pigs. The PROK1-stimulated proliferation of primary porcine endometrial endothelial (PEE) cells involved PI3K/AKT/mTOR, MAPK, cAMP, and NFKB signaling pathways. Furthermore, PROK1 via PROKR1 promoted the formation of capillary-like structures by PEE cells. PROK1 also stimulated VEGFA and PGF2α secretion, which in turn may indirectly support angiogenic changes within endometrial tissue. In summary, our study suggests that PROK1 acts as an embryonic signal mediator that regulates endometrial angiogenesis and secretory function during the implantation and early placentation period in pigs.

Estradiol-17ß-Induced Changes in the Porcine Endometrial Transcriptome In Vivo.

Estradiol-17ß (E2) is a key hormone regulating reproductive functions in females. In pigs, E2, as the main conceptus signal, initiates processes resulting in prolonged corpus luteum function, embryo development, and implantation. During early pregnancy the endometrium undergoes morphological and physiological transitions that are tightly related to transcriptome changes. Recently, however, the importance of E2 as a primary conceptus signal in the pig has been questionable. Thus, the aim of the present study was to determine the effects of E2 on the porcine endometrial transcriptome in vivo and to compare these effects with transcriptome profiles on day 12 of pregnancy. Microarray analysis revealed differentially expressed genes (DEGs) in response to E2 with overrepresented functional terms related to secretive functions, extracellular vesicles, cell adhesion, proliferation and differentiation, tissue rearrangements, immune response, lipid metabolism, and many others. Numerous common DEGs and processes for the endometrium on day 12 of pregnancy and E2-treated endometrium were identified. In summary, the present study is the first evidence for the effect of E2 on transcriptome profiles in porcine endometrium in vivo in the period corresponding to the maternal recognition of pregnancy. The presented results provide a valuable resource for further targeted studies considering genes and pathways regulated by conceptus-derived estrogens and their role in pregnancy establishment.

Prostaglandin F2α promotes embryo implantation and development in the pig.

Successful establishment and development of pregnancy requires proper communication between developing conceptuses and the maternal reproductive tract. Prostaglandins are key players involved in the regulation of reproductive processes in mammals including pigs. Due to its luteolytic action, prostaglandin F2-alpha (PGF2α) is mainly considered as an undesirable factor during early pregnancy. However, its content in the uterine lumen is elevated in pigs and other mammals. Recently, we reported an important role of PGF2α in the endometrium during early pregnancy in the pig. Thus, the aim of the present study was to determine whether PGF2α can act on porcine trophoblast and if so, to elucidate what effect it could exert. We detected increased expression of PGF2α receptor during the implantation period (from day 14 until day 19 of pregnancy). Global gene expression profiling using microarrays and quantitative PCR studies revealed that PGF2α acting on porcine trophoblast cells in vitro alters expression of genes potentially involved in processes related to implantation, such as: cell proliferation, focal adhesion, extracellular matrix binding, cell migration, cytoskeleton organization, immune interactions, ion homeostasis, and lipid metabolism. Using primary porcine trophoblast cells, we demonstrated that PGF2α stimulated trophoblast cell proliferation and adhesion to extracellular matrix protein. This was likely mediated by mitogen-activated protein kinases (MAPK1/3) and focal adhesion kinase (FAK) since we observed increased phosphorylation of MAPK1/3 and FAK in trophoblast cells treated with PGF2α. To conclude, the present report indicates a novel role for PGF2α in the porcine conceptus as a para- and autocrine factor supporting pregnancy establishment.

Estimating acute and chronic exposure of children and adults to chlorpyrifos in fruit and vegetables based on the new, lower toxicology data.

This paper presents, for the first time, results for chlorpyrifos (CHLP) in Polish fruits and vegetables over the course of a long period of research, 2007-2016, with toxicological aspects. The challenge of this study was to re-evaluate the impact of chlorpyrifos residues in fruit and vegetables on health risk assessed via acute and chronic exposure based on old and new, lower, established values of: Average Daily Intakes (ADIs)/Acute Reference Doses (ARfDs) and Maximum Residue Levels (MRLs). A total of 3 530 samples were collected, and CHLP in the range of 0.005-1.514 mg/kg was present in 10.2% of all samples. The MRL was exceeded in 0.7% of all samples (MRL established in 2009-2015), and recalculation yielded a much greater number of violations for the new MRL (2016), which exceeded 2.9% of all samples. Acute exposure to CHLP calculated according to the old, higher toxicological data (0.10 mg/kg bw/day), does not exceed 14% of its respective ARfDs for adults and both groups of children, but when calculated for incidental cases according to the current value (ARfD 0.005 mg/kg bw) for infants and toddlers, was above 100% of its respective ARfDs in: white cabbage (263.65% and 108.24%), broccoli (216.80% and 194.72%) and apples (153.20% and 167.70%). The chronic exposure calculated for both newly established ADI values (0.001 mg/kg bw/day and 0.100 mg/kg bw/day) appears to be relatively low for adults and children.

The Study of Anti-/Pro-Oxidant, Lipophilic, Microbial and Spectroscopic Properties of New Alkali Metal Salts of 5-O-Caffeoylquinic Acid.

Lithium, sodium, potassium, rubidium and caesium salts of 5-O-caffeoylquinic acid (chlorogenic acid, 5-CQA) were synthesized and described by FT-IR (infrared spectroscopy), FT-Raman (Raman spectroscopy), UV (UV absorption spectroscopy), ¹H (400.15 MHz), 13C (100.63 MHz) NMR (nuclear magnetic resonance spectroscopy). The quantum-chemical calculations at the B3LYP/6-311++G** level were done in order to obtain the optimal structures, IR spectra, NBO (natural bond orbital) atomic charges, HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) orbitals and chemical reactivity parameters for 5-CQA and Li, Na and K 5-CQAs (chlorogenates). The DPPH (α, α-diphenyl-ß-picrylhydrazyl) and FRAP (ferric reducing antioxidant power) assays were used for the preliminary estimation of the antioxidant properties of alkali metal chlorogenates and chlorogenic acid. In the DPPH assay the EC50 parameter were equal to 7.39 µM for 5-CQA and was in the range of 4.50-5.89 µM for salts. The FRAP values for two different concentrations (5 and 2.5 µM) of the studied compounds were respectively 114.22 and 72.53 µM Fe2+ for 5-CQA, whereas for salts they were 106.92-141.13 and 78.93-132.00 µM Fe2+. The 5-CQA and its alkali metal salts possess higher antioxidant properties than commonly applied antioxidants (BHA, BHT, l-ascorbic acid). The pro-oxidant action of these compounds on trolox oxidation was studied in the range of their concentration 0.05-0.35 µM. The lipophilicity (logkw) of chlorogenates and chlorogenic acid was determined by RP-HPLC (reverse phase-high performance liquid chromatography) using five different columns (C8, PHE (phenyl), CN (cyano), C18, IAM (immobilized artificial membrane)). The compounds were screened for their in vitro antibacterial activity against E. coli, Bacillus sp., Staphylococcus sp., Streptococcus pyogenes and antifungal activity against Candida sp. The 5-CQA possessed lower antibacterial (minimal inhibitory concentration, MIC = 7.06 mM) and antifungal (MIC = 14.11 mM) properties than its alkali metal salts (MIC values: 6.46-2.63 mM and 12.91-5.27mM, respectively). The synthesized chlorogenates possessed better antioxidant, lipophilic, antimicrobial as well as lower pro-oxidant properties than the ligand alone. Moreover, a systematic change of the activity of alkali metal salts along the series Li→Cs suggests that there are correlations between the studied biological properties. The type of metal cation in the carboxylate group of chlorogenate is crucial for the activity of studied compounds.

Embryo-maternal dialogue during pregnancy establishment and implantation in the pig.

Porcine conceptuses secrete pregnancy-recognition signals (estrogens, including estradiol-17ß) that inhibit luteolysis, thereby prolonging progesterone production by corpora lutea. The supportive mechanism by which the conceptus also inhibits luteolysis is by shifting endometrial prostaglandin (PG) synthesis to luteoprotective PGE2. Progesterone stimulates endometrial production of factors that are essential for conceptus development. Priming the uterus by progesterone and loss of progesterone receptors from the uterine epithelium by D1ay 10-12 after estrus are key for achieving endometrial receptivity for implantation. Conceptus implantation involves a series of events, many resembling the inflammatory reaction, that are greatly influenced by cytokines, growth factors, and prostaglandins. We herein present a novel, dual role for PGF2α in corpora lutea that depends on the acquisition of luteolytic sensitivity, based on the knowledge that PGF2α triggers pathways involved in luteolysis during the estrous cycle or/and may have an alternative function in maintaining progesterone synthesis during pregnancy. We also point out a new role for PGF2α that, together with PGE2, can act as embryonic signal mediators. PGF2α, which until recently was considered undesirable for promoting pregnancy, is now known to stimulate conceptus-maternal interactions and angiogenesis in the endometrium. This function is in line with other important prostaglandin functions, such as stimulating adhesion of trophoblasts (PGE2, PGI2) as well as endometrial vascular functions and trophoblast cell proliferation (PGI2). Finally, microRNAs have emerged as important post-transcriptional regulators of gene function, adding a new area of investigation that may enhance understanding of conceptus-endometrial interactions.

Evolution of novel sorbents for effective clean-up of honeybee matrix in highly toxic insecticide LC/MS/MS analysis.

Highly toxic insecticides (HTIs) belonging to different chemical groups are dangerous to pollinating organisms, even in sublethal doses. An important objective of this study was to develop a method to determine over fifty HTIs at very low concentrations in the bee matrix. The novelty of this research involved obtaining extract completely free from beewaxes, lipids and proteins using EMR-lipid (enhanced matrix removal-lipid), chitin and Z-Sep+ (zirconium oxide and C18 dual-bonded to silica) as clean-up sorbents. Different parameters, such as weight of bees, extraction solvent, and freezing time were evaluated. Determinations were made using liquid chromatography-tandem mass spectrometry (LC-MS/MS). EMR-lipid allows for removing most of the fatty co-extracts and increases the overall performance of the method by reducing the matrix effects (ME) without significant analyte loss. The established modified QuEChERS method based on 1% acetic acid in acetonitrile extraction followed by EMR-lipid clean-up was validated at three different spiking levels (0.001, 0.01 and 0.1mgkg-1). Precision, calculated as relative standard deviation (RSD), was below 20%. The proposed method was used to determine sublethal doses of these insecticides in real samples of dead honeybees.

Multiclass pesticide residue analysis in fish muscle and liver on one-step extraction-cleanup strategy coupled with liquid chromatography tandem mass spectrometry.

The analysis of pesticide residues in fish samples is challenging due to the low concentrations and large number of analytes that need to be monitored and quantified in a complex matrix. This is the first report providing a novel one-step extraction-cleanup strategy for simultaneous analysis of over 340 pesticides in a fatty fish and liver matrix, coupled with liquid-chromatography tandem mass spectrometry. The samples of fish muscle and liver were prepared according to the modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure, wherein the extraction and cleanup protocol were integrated into one step. Among the tested cleanup dispersive solid phase extraction sorbents (C18, primary-secondary amine, Z-Sep), chitin yielded the best results. Spike-in experiments were carried out at three different spiking levels in fish and liver to determine the recovery, precision and limits of detection of the method as well as the matrix effect. The method's detection limits ranged from 0.05 to 1.2µgkg-1, while recoveries of most pesticides were in the range of 70-120% with associated precision - relative standard deviations below 20%. A linear relationship was observed within the range of 0.005-1mgkg-1, and the correlation coefficient was R2>0.997. Expanded measurement uncertainty was estimated to be between 7% and 52%, on average. Matrix effects were evaluated and were not significant for the vast majority of pesticides. The validated method was employed in the analysis of 54 real fish and liver samples in which 10 different pesticides with concentrations ranging from 0.005 to 0.047mgkg-1 were detected.

Dissipation of S-metolachlor in plant and soil and effect on enzymatic activities.

The present study aimed at evaluating the dissipation of S-metolachlor (S-MET) at three doses in maize growing on diverse physico-chemical properties of soil. The effect of herbicide on dehydrogenase (DHA) and acid phosphatase (ACP) activity was estimated. A modified QuEChERS method using LC-MS/MS has been developed. The limit of quantification (0.001 mg kg-1) and detection (0.0005 mg kg-1) were very low for soil and maize samples. The mean recoveries and RSDs for the six spiked levels (0.001-0.5 mg kg-1) were 91.3 and 5.8%. The biggest differences in concentration of S-MET in maize were observed between the 28th and 63rd days. The dissipation of S-MET in the alkaline soil was the slowest between the 2nd and 7th days, and in the acidic soil between the 5th and 11th days. DT50 of S-MET calculated according to the first-order kinetics model was 11.1-14.7 days (soil) and 9.6-13.9 days (maize). The enzymatic activity of soil was higher in the acidic environment. One observed the significant positive correlation of ACP with pH of soil and contents of potassium and magnesium and negative with contents of phosphorus and organic carbon. The results indicated that at harvest time, the residues of S-MET in maize were well below the safety limit for maize. The findings of this study will foster the research on main parameters influencing the dissipation in maize ecosystems.

Prostaglandin F2α promotes angiogenesis and embryo-maternal interactions during implantation.

Implantation in humans and other mammals is a critical period during which high embryonic mortality rates occur. Prostaglandins (PGs) are key mediators regulating interactions between the reproductive tract and the conceptus (embryo with extraembryonic membranes). Although the significance of PGF2α as a regulator of corpus luteum regression is well established, the role of its high amounts in the uterine lumen in most mammals, regardless of placentation type, during the implantation period remains unresolved. We hypothesized that PGF2α acting as an embryonic signal mediator contributes to pregnancy establishment. Using a porcine model, we demonstrated that the conceptus and its signal (estradiol-17ß) elevated endometrial expression of PGF2α receptor (PTGFR) in vivo and in vitro PTGFR protein was expressed mainly in luminal epithelial (LE) and glandular epithelial cells and blood vessels in the endometrium. PGF2α stimulated the MAPK1/3 pathway in endometrial LE cells that coincided with elevated gene expression and secretion of endometrial vascular endothelial growth factor A (VEGFA) protein. PGF2α-PTGFR and adenylyl cyclase signaling were involved in this process. PGF2α-induced VEGFA acting through its receptors stimulated proliferation of endometrial endothelial cells. Moreover, PGF2α elevated gene expression of biglycan, matrix metalloproteinase 9, transforming growth factor ß3, and interleukin 1α in the endometrium. In summary, our study indicates that PGF2α participates in pregnancy establishment by promoting angiogenesis and expression of genes involved in tissue remodeling and conceptus-maternal interactions in porcine endometrium during early pregnancy.

Removal of 16 pesticide residues from strawberries by washing with tap and ozone water, ultrasonic cleaning and boiling.

The effects of washing with tap and ozone water, ultrasonic cleaning and boiling on 16 pesticide (ten fungicides and six insecticides) residue levels in raw strawberries were investigated at different processing times (1, 2 and 5 min). An analysis of these pesticides was conducted using gas chromatography with nitrogen-phosphorous and electron capture detection (GC-NPD/ECD). The processing factor (PF) for each pesticide in each processing technique was determined. Washing with ozonated water was demonstrated to be more effective (reduction from 36.1 to 75.1 %) than washing with tap water (reduction from 19.8 to 68.1 %). Boiling decreased the residues of the most compounds, with reductions ranging from 42.8 to 92.9 %. Ultrasonic cleaning lowered residues for all analysed pesticides with removal of up to 91.2 %. The data indicated that ultrasonic cleaning and boiling were the most effective treatments for the reduction of 16 pesticide residues in raw strawberries, resulting in a lower health risk exposure. Calculated PFs for alpha-cypermethrin were used to perform an acute risk assessment of dietary exposure. To investigate the relationship between the levels of 16 pesticides in strawberry samples and their physicochemical properties, a principal component analysis (PCA) was performed. Graphical abstract ᅟ.

The influence of effective microorganisms (EM) and yeast on the degradation of strobilurins and carboxamides in leafy vegetables monitored by LC-MS/MS and health risk assessment.

The aim of this study was to determine the behaviour of strobilurin and carbocyamides commonly used in chemical protection of lettuce depending on carefully selected effective microorganisms (EM) and yeast (Y). Additionally, the assessment of the chronic health risk during a 2-week experiment was performed. The statistical method for correlation of physico-chemical parameters and time of degradation for pesticides was applied. In this study, the concentration of azoxystrobin, boscalid, pyraclostrobin and iprodione using liquid chromatography-mass spectrometry (LC-MS/MS) in the matrix of lettuce plants was performed, and there was no case of concentration above maximum residues levels. Before harvest, four fungicides and their mixture with EM (1 % and 10 %) and/or yeast 5 % were applied. In our work, the mixtures of 1%EM + Y and 10%EM + Y both stimulated and inhibited the degradation of the tested active substances. Adding 10%EM to the test substances strongly inhibited the degradation of iprodione, and its concentration decreased by 30 %, and in the case of other test substances, the degradation was approximately 60 %. Moreover, the addition of yeast stimulated the distribution of pyraclostrobin and boscalid in lettuce leaves. The risk assessment for the pesticides ranged from 0.4 to 64.8 % on day 1, but after 14 days, it ranged from 0.0 to 20.9 % for children and adults, respectively. It indicated no risk of adverse effects following exposure to individual pesticides and their mixtures with EM and yeast.

Studies of pesticide residues in tomatoes and cucumbers from Kazakhstan and the associated health risks.

The aim of the present study was to assess the level of pesticide residues in vegetables in the Almaty Region of Kazakhstan and to determine the potential health risks associated with the exposures to these pesticides. A total of 82 samples of cucumbers and tomatoes from top agro-based market and greenhouses were analysed using a gas chromatography-micro electron capture detector/nitrogen-phosphorous detector (GC-µECD/NPD), a multiresidue method to analyse 184 different pesticide types. The results indicated that more than half of samples (59 %) contained 29 pesticides, in which 10 are not registered in Kazakhstan, ranging from 0.01 to 0.88 mg kg(-1), and 28 % contained pesticide residues above maximum residue levels (MRLs). The estimated daily intakes (EDIs) ranged from 0.01 % of the acceptable daily intake (ADI) for pyrimethanil to 12.05 % of the ADI for lambda-cyhalothrin. The most critical commodity is triazophos and flusilazole in tomatoes, contributing 70.8 and 42.5 % to the acute hazard index (aHI). The results provided important information on the current pesticide contamination status of two commonly consumed vegetables and pointed an urgent need to control the use of plant protection products applied, especially potentially persistent pesticides, such as endosulfan and dicofol. These results also show that the detected pesticides may be considered a public health problem.

The regulation of the expression of prokineticin 1 and its receptors and its mechanism of action in the porcine corpus luteum.

Prokineticin 1 (PROK1) is an important factor in pregnancy establishment in pigs, acting at the embryo-maternal interface and the corpus luteum (CL). Estradiol-17ß (E2) is the primary pregnancy recognition signal in pigs, and its effects are augmented by luteotropic prostaglandin E2 (PGE2). On the contrary, prostaglandin F2α (PGF2α) exerts mainly a luteolytic effect. The present study aimed to elucidate whether E2, PGE2, and PGF2α regulate the expression of PROK1 and its receptors in the porcine CL and to determine the PROK1 effect on luteal endothelial cells and pathways that may be involved in this regulation. The effects of E2, PGE2, and PGF2α on the expressions of PROK1 and its receptors in the CL were studied using an in vitro model of ultrathin luteal tissue explants model. Additionally, the effects of E2 and PGE2 on the PROK1 system were determined using an in vivo approach, in which the hormones were administered into the uterine lumen to imitate their secretion by embryos. Endothelial cell proliferation was measured using the colorimetric method. E2 acting via estrogen receptors simulated the mRNA and protein expressions of PROK1 and PROKR1 in CL explants in vitro (p < 0.05). The simultaneous action of E2 with PGE2 enhanced the expression of luteal PROK1 mRNA in vitro (p < 0.05). Estradiol-17ß acting alone significantly increased PROK1 mRNA levels in vivo, whereas E2 simultaneously administered with PGE2 significantly elevated the PROK1 mRNA expression and PROKR1 mRNA and protein contents in CLs adjacent to uterine horns receiving hormonal infusion compared with CLs adjacent to placebo-treated uterine horns (p < 0.01). The PROK1 protein expression was significantly higher in the CLs of pigs treated with E2, PGE2, and E2 together with PGE2 than in the control group. PGF2α increased the PROK1 mRNA content in CLs on days 12 and 14 of the estrous cycle (p < 0.05). The expression of PROKR2 at the mRNA and protein levels remained unchanged in response to in vitro and in vivo treatments. PROK1 stimulated the proliferation of luteal endothelial cells by activating the MAPK, AKT, and mTOR pathways (p < 0.05). In summary, the luteal expressions of PROK1 and PROKR1 in early pregnancy are regulated by E2 and PGE2. PROK1 stimulates luteal angiogenesis by activating the MAPK, AKT, and mTOR pathways. The regulation of luteal PROK1 expression by PGF2α indicates PROK1's putative role during luteolysis. We conclude that PROK1-PROKR1 signaling supports luteal function during CL rescue in pregnancy in pigs.