Zita Zrínyi

Flavonoid diosmetin increases ATP levels in kidney cells and relieves ATP depleting effect of ochratoxin A

Diosmetin (DIOS) is a flavone aglycone commonly occurring in citrus species and olive leaves, in addition it is one of the active ingredients of some medications. Based on both in vitro and in vivo studies several beneficial effects are attributed to DIOS but the biochemical background of its action seems to be complex and it has not been completely explored yet. Previous investigations suggest that most of the flavonoid aglycones have negative effect on ATP synthesis in a dose dependent manner. In our study 17 flavonoids were tested and interestingly DIOS caused a significant elevation of intracellular ATP levels after 6- and 12-h incubation in MDCK kidney cells. In order to understand the mechanism of action, intracellular ATP and protein levels, ATP/ADP ratio, cell viability and ROS levels were determined after DIOS treatment. In addition, impacts of different enzyme inhibitors and effect of DIOS on isolated rat liver mitochondria were also tested. Finally, the influence of DIOS on the ATP depleting effect of the mycotoxin, ochratoxin A was also investigated. Our major conclusions are the followings: DIOS increases intracellular ATP levels both in kidney and in liver cells. Inhibition of glycolysis or citric acid cycle does not decrease the observed effect. DIOS-induced elevation of ATP levels is completely abolished by the inhibition of ATP synthase. DIOS is able to completely reverse the ATP-depleting effect of the mycotoxin, ochratoxin A. Most probably the DIOS-induced impact on ATP system does not originate from the antioxidant property of DIOS. Based on our findings DIOS may be promising agent to positively influence ATP depletion caused by some metabolic poisons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

ABSTRACT Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinsons disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinsons disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP. Summary: PACAP has a neuroprotective effect in different toxin-induced rat and snail parkinsonian models, acting partially through the same mechanisms.

Noninvasive embryo viability assessment by quantitation of human haptoglobin alpha-1 fragment in the in vitro fertilization culture medium: an additional tool to increase success rate

OBJECTIVEnTo find new candidate molecules to assess embryo viability in a noninvasive manner.nnnDESIGNnProspective, blinded study with randomized sample collection.nnnSETTINGnUniversity research center.nnnPATIENTS(S)nNinety embryos implanted in 53 randomly selected patients (mean ± SD age, 32.3 ± 5.1xa0years) were analyzed.nnnINTERVENTION(S)nSuperovulation treatment was initiated by the administration of the GnRh agonist triptorelin and individual dosages of recombinant FSH. Ovulation was induced by the injection of hCG. Oocytes were fertilized by intracytoplasmic sperm injection.nnnMAIN OUTCOME MEASURE(S)nLiquid chromatography coupled mass spectrometric quantification of the α-1 fragment of human haptoglobin in the culture medium.nnnRESULT(S)nA novel polypeptide marker was found that might be helpful to differentiate between potentially viable and nonviable embryos. This molecule was identified with tandem mass spectrometry as the α-1 fragment of human haptoglobin. Significant correlation was found in the amount of the peptide fragment and the outcome of pregnancy. In the culture media of embryos that were assigned in the biochemical assay as nonviable (according to the amount of the haptoglobin fragment), there were no pregnancies detected; this assay revealed a 100% successful selection of the nonviable embryos. In the group assigned as viable, the rate of pregnancy was 54.7%.nnnCONCLUSION(S)nViability of the embryo during the IVF process is assessed by microscopic inspection, resulting in a pregnancy rate of 25%-30%. Detection and quantitation of the α-1 haptoglobin fragment of the culture medium proved to be a useful additional method for identifying nonviable embryos, increasing the success rate to 50%.

HPLC-MS/MS analysis of steroid hormones in environmental water samples

Today, freshwaters, such as lakes and rivers, are subject to controlled pollution. Steroid hormones are chemically very stable highly lipophilic molecules. Their biological properties have a strong impact on the endocrine regulation of species. Steroids have estrogenic, androgenic, thyroidogenic or progestogenic effects and based on them, they could disturb the physiological mechanisms of freshwater species. We focused on progestins as they are the main active ingredients of contraceptive pharmaceuticals. Progestins have been shown to impair reproduction in fish, amphibians, and mollusks at low ng/L concentrations. Certain progestins, such as levonorgestrel (LNG) have androgenic properties also. We selected the most used active substances drospirenone (DRO), LNG, and progesterone (PRG) and then developed and optimized a liquid chromatographic-mass spectrometric method with solid-phase extraction to measure them. Using our sensitive method (LOQ 0.03-0.11u2009ng/L) we could measure steroids even between 0.1 and 1u2009ng/L. Analyzing freshwater samples from the Lake Balaton catchment area, we found influents where the concentration of these hormones was 0.26-4.30 (DRO), 0.85-3.40 (LNG), and 0.23-13.67 (PRG) ng/L. Out of 53 collecting places, 21 contained measurable progestin levels, which clearly demonstrates the applicability of our method, legitimates toxicology experiments with effected species, and indicates monitoring efforts.

β-Estradiol and ethinyl-estradiol contamination in the rivers of the Carpathian Basin

Abstract17β-Estradiol (E2) and 17α-ethinyl estradiol (EE2), which are environmental estrogens, have been determined with LC-MS in freshwater. Their sensitive analysis needs derivatization and therefore is very hard to achieve in multiresidue screening. We analyzed samples from all the large and some small rivers (River Danube, Drava, Mur, Sava, Tisza, and Zala) of the Carpathian Basin and from Lake Balaton. Freshwater was extracted on solid phase and derivatized using dansyl chloride. Separation was performed on a Kinetex XB-C18 column. Detection was achieved with a benchtop orbitrap mass spectrometer using targeted MS analysis for quantification. Limits of quantification were 0.05xa0ng/L (MS1) and 0.1xa0ng/L (MS/MS) for E2, and 0.001xa0ng/L (MS1) and 0.2xa0ng/L (MS/MS) for EE2. River samples contained n.d.–5.2xa0ng/L E2 and n.d.–0.68xa0ng/L EE2. Average levels of E2 and EE2 were 0.61 and 0.084xa0ng/L, respectively, in rivers, water courses, and Lake Balaton together, but not counting city canal water. EE2 was less abundant, but it was still present in almost all of the samples. In beach water samples from Lake Balaton, we measured 0.076–0.233 E2 and n.d.–0.133 EE2. A relative high amount of EE2 was found in river Zala (0.68xa0ng/L) and in Hévíz-Páhoki canal (0.52xa0ng/L), which are both in the catchment area of Lake Balaton (Hungary).

Structure related effects of flavonoid aglycones on cell cycle progression of HepG2 cells: Metabolic activation of fisetin and quercetin by catechol-O-methyltransferase (COMT)

Dietary flavonoids are abundant in the Plant Kingdom and they are extensively studied because of their manifold pharmacological activities. Recent studies highlighted that cell cycle arrest plays a key role in their antiproliferative effect in different tumor cells. However, structure-activity relationship of flavonoids is poorly characterized. In our study the influence of 18 flavonoid aglycones (as well as two metabolites) on cell cycle distribution was investigated. Since flavonoids are extensively metabolized by liver cells, HepG2 tumor cell line was applied, considering the potential metabolic activation/inactivation of flavonoids. Our major observations are the followings: (1) Among the tested compounds diosmetin, fisetin, apigenin, lutelin, and quercetin provoked spectacular extent of G2/M phase cell cycle arrest. (2) Inhibition of catechol-O-methyltransferase enzyme by entacapone decreased the antiproliferative effects of fisetin and quercetin. (3) Geraldol and isorhamnetin (3-O-methylated metabolites of fisetin and quercetin, respectively) demonstrated significantly higher antiproliferative effect on HepG2 cells compared to the parent compounds. Based on these results, O-methylated flavonoid metabolites or their chemically modified derivatives may be suitable candidates of tumor therapy in the future.

Lithium Induces ER Stress and N-Glycan Modification in Galactose-Grown Jurkat Cells

We previously reported that lithium had a significant impact on Ca2+ regulation and induced unfolded protein response (UPR) in yeast cells grown on galactose due to inhibition of phosphoglucomutase (PGM), however the exact mechanism has not been established yet. In this study, we analysed lithiums effect in galactose-fed cells to clarify whether these ER-related changes are the result of a relative hypoglycemic state. Furthermore, we investigated whether the alterations in galactose metabolism impact protein post-translational modifications. Thus, Jurkat cells were incubated in glucose or galactose containing media with or without lithium treatment. We found that galactose-fed and lithium treated cells showed better survivability than fasting cells. We also found higher UDP-Hexose and glycogen levels in these cells compared to fasting cells. On the other hand, the UPR (X-box binding protein 1 mRNA levels) of galactose-fed and lithium treated cells was even greater than in fasting cells. We also found increased amount of proteins that contained N-linked N-acetyl-glucosamine, similar to what was reported in fasting cells by a recent study. Our results demonstrate that lithium treatment of galactose-fed cells can induce stress responses similar to hypoglycemia, however cell survival is still secured by alternative pathways. We propose that clarifying this process might be an important addition toward the better understanding of the molecular mechanisms that regulate ER-associated stress response.

Effect of progesterone and its synthetic analogs on reproduction and embryonic development of a freshwater invertebrate model

The presence of a mixture of progestogens at ng/L concentration levels in surface waters is a worldwide problem. Only a few studies explore the effect of progestogen treatment in a mixture as opposed to individual chemicals to shed light on how non-target species respond to these contaminants. In the present study, we used an invertebrate model species, Lymnaea stagnalis, exposed to a mixture of four progestogens (progesterone, levonorgestrel, drospirenone, and gestodene) in 10ng/L concentration for 3 weeks. Data at both physiological and cellular/molecular level were analyzed using the ELISA technique, stereomicroscopy combined with time lapse software, and capillary microsampling combined with mass spectrometry. The treatment of adult Lymnaeas caused reduced egg production, and low quality egg mass on the first week, compared to the control. Starting from the second week, the egg production, and the quality of egg mass were similar in both groups. At the end of the third week, the egg production and the vitellogenin-like protein content of the hepatopancreas were significantly elevated in the treated group. At the cellular level, accelerated cell proliferation was observed during early embryogenesis in the treated group. The investigation of metabolomic changes resulted significantly elevated hexose utilization in the single-cell zygote cytoplasm, and elevated adenylate energy charge in the egg albumen. These changes suggested that treated snails provided more hexose in the eggs in order to improve offspring viability. Our study contributes to the knowledge of physiological effect of equi-concentration progestogen mixture at environmentally relevant dose on non-target aquatic species.

Complex molecular changes induced by chronic progestogens exposure in roach, Rutilus rutilus

In our previous study, we measured 0.23-13.67ng/L progestogens (progesterone, drospirenone, levonorgestrel) in natural waters in the catchment area of the largest shallow lake of Central Europe, Lake Balaton. Progestogen contaminations act as potent steroids with mixed progestagenic, androgenic and mild estrogenic effects that is why our aim was to investigate the morphological and molecular effects of mixture of progesterone, drospirenone, and levonorgestrel in environmentally relevant (10ng/L) and higher (50 and 500ng/L) exposure concentrations in common roach, Rutilus rutilus. Steroids (e.g. progestogens) and the protein deglycase DJ-1 chaperon molecule aim the same target molecules in cells, therefore, we hypothesized that a relationship may exist between progestogens and DJ-1. Furthermore, our other aim was to follow the changes of signal molecules of different biological function due to progestogen treatment in serum and brain. Adult roaches were exposed to 10, 50 and 500ng/L of mixture of progestogen for 42 days and their somatic indices (brain-somatic, liver-somatic, gonadosomatic and kidney-somatic) were measured. Vitellogenin (VTG) expression (estrogen effect) or inhibition (androgen effect) in fish is a widely used biomarker so we measured its changes in liver by ELISA. To determine the quantity and to map the spatial distribution of DJ-1 chaperon protein the brain and liver tissues were analyzed by ELISA and immunohistochemistry. Furthermore, we also studied molecular alterations: a) in the serum by measuring cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride concentrations and b) in brain homogenate using a cell stress array kit (26 protein). The somatic index of liver and kidney significantly in all the treated groups, whereas the gonadosomatic index of 500ng/L treated group showed significant decrease compared to control animals. VTG level increased significantly in 500ng/L progestogen treated group. Since the concentration of DJ-1 significantly increased in brain and liver in all progestogen treatment groups, the DJ-1 protein could be able to a more sensitive marker than VTG. Serum LDL and cholesterol levels of exposed fish were significantly decreased. DJ-1 was mediated through the stimulation of the expression of LDL-receptor which facilitates reuptake subsequently. In summary, our observations unfolded new data about molecular alterations induced by the combined action of environmental progestogens. In addition, the DJ-1 chaperon protein as a possible biomarker helped to trace the abiotic chemical environmental contaminations, like progestogens in the freshwater ecosystems.

Time courses of changes of para-, meta-, and ortho-tyrosine in septic patients: A pilot study

Objectives: Sepsis is associated with oxidative stress. Due to oxidative stress, three tyrosine isoforms, para-, meta-, and ortho-tyrosine (p-, m-, and o-Tyr), can be formed non-enzymatically in smaller amounts. p-Tyr is mainly formed physiologically in the kidneys through the activity of the phenylalanine hydroxylase enzyme. The three tyrosine isoforms may undergo different renal handling. Methods: Twenty septic patients were involved in the study and 25 healthy individuals served as controls. Blood and urine levels of p-, m-, and o-Tyr were measured on admission and four consecutive days. Results: Serum m-Tyr levels were higher in septic patients than in controls on days 2 (P = 0.031) and 3 (P = 0.035). Serum p-Tyr levels were lower in the cases than in controls on days 1 (P = 0.005) and 2 (P = 0.040), and subsequently normalized due to a day-by-day elevation (P = 0.002). The tendency of urinary m-Tyr concentration was decreasing (P = 0.041), while that of urinary p-Tyr concentration was increasing (P = 0.001). Fractional excretion of m-Tyr (FEm-Tyr) showed a decreasing tendency (P = 0.009), and was, on all days, higher than FEp-Tyr, which remained near-normal, less than 4%. Procalcitonin showed significant correlation with FEm-Tyr (r = 0.454; P < 0.001). Discussion: Our data suggest that the oxidative stress marker m-Tyr and physiologic p-Tyr may be handled differently in septic patients. The excretion of m-Tyr correlates with inflammation. m-Tyr may be actively secreted or produced in the kidney in some patients, whereas the decreased serum level of p-Tyr is a consequence of diminished renal production and not of renal loss.