Propylparaben-induced disruption of energy metabolism in human HepG2 cell line leads to increased synthesis of superoxide anions and apoptosis.

The effect of propylparaben (in final concentrations 0.4 ng/ml, 2.3 ng/ml and 4.6 ng/ml) on the energy metabolism of HepG2 hepatocytes, superoxide anion synthesis, apoptosis and necrosis is described. Propylparaben can be toxic to liver cells due to the increased production of superoxide anions, which can contribute to a reduced concentration of superoxide dismutase in vivo and impairment of the body's antioxidant mechanisms. Finally, a further reduction in the mitochondrial membrane potential and uncoupling of the respiratory chain resulting in a reduction in ATP concentration as a result of mitochondrial damage may lead to cell death by apoptosis.

Fluoride as a factor initiating and potentiating inflammation in THP1 differentiated monocytes/macrophages.

It is well known that exposure to fluorides lead to an increased ROS production and enhances the inflammatory reactions. Therefore we decided to examine whether cyclooxygenases (particular COX-2) activity and expression may be changed by fluoride in THP1 macrophages and in this way may change the prostanoids biosynthesis. In the present work we demonstrate that fluoride increased concentration of PGE2 and TXA2 in THP1 macrophages. Following exposure to 1-10 µM NaF, COX-2 protein and COX-2 transcript increased markedly. COX-2 protein up-regulation probably is mediated by ROS, produced during fluoride-induced inflammatory reactions. Additional fluoride activates the transcription factor, nuclear factor (NF)-kappaB, which is involved in the up-regulation of COX-2 gene expression. This study indicated that even in small concentrations fluoride changes the amounts and activity of COX-1 and COX-2 enzymes taking part in the initiating and development of inflammatory process.

Hymenolepis diminuta: analysis of the expression of Toll-like receptor genes (TLR2 and TLR4) in the small and large intestines of rats. Part II.

Toll-like receptors in the gastrointestinal tract can influence intestinal homeostasis and play a role in the repair and restitution of intestinal epithelium following tissue damage. In our previous study a statistically significant increase in the level of TLR4 and TLR2 gene expression was observed in rats in early stages of hymenolepidosis. Moreover, the immunopositive cell number and the intensity of immunohistochemical staining (indicating the presence of TLRs within intestinal epithelial cells) increased over the infection period. In this paper, we determined changes in the expression of TLR2 and TLR4 and the number of anaerobic intestinal commensal bacteria in Hymenolepis diminuta infected rats. In the isolated jejunum of infected rats at 16 days post infection (dpi), the expression of TLR4 and TLR2 was significantly higher than uninfected rats. In the colon, a statistically significantly increased expression of TLR2 was observed from 16 to 40 dpi, and TLR4 from 16 to 60 dpi. The jejunum and colon of infected rats contained Gram-negative bacteria (Escherichia coli), Gram-positive bacteria (Enterococcus, Streptococcus, Staphylococcus, Bacillus, Lactobacillus) and Candida. The total number of intestinal bacteria was higher in H. diminuta infected rats, but the observed microbiota had only minor effects on the expression of TLR2 and TLR4. Toll-like receptors play a role in maintaining epithelial barrier function in response to enteric pathogens and parasites. In our study, the alteration of TLR2 and TLR4 expression in the infected rats indicates the potential role of the innate immune system in the pathomechanism of this infection.

Perinatal exposure to lead induces morphological, ultrastructural and molecular alterations in the hippocampus.

The aim of this paper is to examine if pre- and neonatal exposure to lead (Pb) may intensify or inhibit apoptosis or necroptosis in the developing rat brain. Pregnant experimental females received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring; the control group received distilled water. During the feeding of pups, mothers from the experimental group were still receiving PbAc. Pups were weaned at postnatal day 21 and the young rats of both groups then received only distilled water until postnatal day 28. This treatment protocol resulted in a concentration of Pb in rat offspring whole blood (Pb-B) below the threshold of 10 µg/dL, considered safe for humans.We studied Casp-3 activity and expression, AIF nuclear translocation, DNA fragmentation, as well as Bax, Bcl-2 mRNA and protein expression as well as BDNF concentration in selected structures of the rat brain: forebrain cortex (FC), cerebellum (C) and hippocampus (H). The microscopic examinations showed alterations in hippocampal neurons.Our data shows that pre- and neonatal exposure of rats to Pb, leading to Pb-B below 10 µg/dL, can decrease the number of hippocampus neurons, occurring concomitantly with ultrastructural alterations in this region. We observed no morphological or molecular features of severe apoptosis or necrosis (no active Casp-3 and AIF translocation to nucleus) in young brains, despite the reduced levels of BDNF. The potential protective factor against apoptosis was probably the decreased Bax/Bcl-2 ratio, which requires further investigation. Our findings contribute to further understanding of the mechanisms underlying Pb neurotoxicity and cognition impairment in a Pb-exposed developing brain.

Hymenolepis diminuta: analysis of the expression of Toll-like receptor genes (TLR2 and TLR4) in the small and large intestines of rats.

Toll receptors play a critical role in the rapid activation of innate immune responses to a variety of pathogens. In mammals, Toll-like receptors (TLR) have been found in both immune related cells and other cells. At present little is known about the participation of TLR in host defense mechanisms during parasitic infections. The aim of this study was to determine the expression of TLR2 and TLR4 genes in rat intestines during experimental hymenolepidosis. There is difference in expression of TLR2 and TLR4 genes in the colon and jejunum in uninfected rats: in the colon, mRNA of the examined TLR is present in much higher amounts than the jejunum, while the protein of the TLR also had a segmented specific distribution. In the jejunum isolated rats infected with Hymeolepis diminuta 6 and 8 days post infection (dpi), mRNA for TLR4 and TLR2 were significantly more strongly expressed in comparison with the uninfected controls. In the colon, a statistically significantly increased expression of TLR4 gene was observed only at 6 dpi, and at 8 dpi for the TLR2 gene. Moreover, we observed that during inflammation, the immunopositive cell number and the intensity of immunohistochemical staining (indicating the presence of TLR within intestinal epithelial cells), increased together with the duration of the infection period.

Altered energy status of primary cerebellar granule neuronal cultures from rats exposed to lead in the pre- and neonatal period.

This paper examines the effect of pre- and neonatal exposure of rats to lead (0.1% lead acetate in drinking water, resulting in rat offspring whole blood lead concentration (Pb-B) 4µg/dL) on the energy status of neuronal mitochondria by measuring changes in ATP, ADP, AMP, adenosine, TAN concentration, adenylate energy charge value (AEC) and mitochondrial membrane potential in primary cerebellar granule neurons (CGC) in dissociated cultures. Fluorescence studies were performed to imaging and evaluate mitochondria mass, mitochondrial membrane potential, intracellular and mitochondrial reactive oxygen species (ROS) production. The Na(+)/K(+) ATPase activity in intact CGC was measured spectrophotometrically. Our data shows that pre- and neonatal exposure of rats to Pb, even below the threshold of whole blood Pb value considered safe for people, affects the energy status of cultured primary cerebellar granule neurons through a decrease in ATP and TAN concentrations and AEC value, inhibition of Na(+)/K(+) ATPase, and increase in intracellular and mitochondrial ROS concentration. These observations suggest that even these low levels of Pb are likely to induce important alterations in neuronal function that could play a role in neurodegeneration.

Disturbances of energetic metabolism in rat epididymal epithelial cells as a consequence of chronic lead intoxication.

Adult male Wistar rats were intoxicated with 1% lead acetate (PbAc) administered in drinking water for nine months, which amounts to a period five times longer than the duration of one spermatogenesis. There were mitochondrial ultrastructure disorders of epididymal epithelial cells observed in PbAc-treated rats; also a significant lead-induced decrease in ATP concentration in epididymal epithelial cells (by 32%, P < 0.05), Adenylate Energy Charge value (AEC) (by 8%, P < 0.05) and an increase in ADP (28.5%, P < 0.05), AMP (27%, P < 0.05) and adenosine (by 56%, P < 0.05). The results were measured using high performance liquid chromatography (HPLC) and detected even at low lead concentrations in whole blood (M:7.03 µg/dL; Q1-Q3: 2.99-7.65). The function of mitochondria in cultured epididymal epithelial cells of control and PbAc-treated animals were evaluated using fluorophores: Mitotracker Green FM and JC-1. After incubation with Mitotracker Green FM, we observed active mitochondria producing bright green fluorescence in the cytoplasm of cultured epididymal epithelial cells, both in the control group and the Pb-treated animals. Incubation of cultured epididymal epithelial cells of animals from both groups produced red-orange fluorescence with the mitochondrial JC-1 probe indicating mitochondria with high membrane potential (ΔΨm > 80-100 mV) and green fluorescence in the mitochondria with low membrane potential (ΔΨm < 80 mV). The results showed that a chronic low-level exposure to lead, even without severe clinical symptoms of contamination, disrupted the ultrastructure and energy metabolism of mitochondria in epididymal epithelial cells.

Expression of E-SOD, GPX5 mRNAs and immunoexpression of Cu/ZnSOD in epididymal epithelial cells of finasteride-treated rats.

We studied the immunoexpression of Cu/Zn superoxide dismutase (Cu/ZnSOD) and mRNAs expression of extracellular superoxide dismutase (E-SOD), and epididymal specific glutathione peroxidase 5 (GPX5), in epithelial cells of caput and cauda epididymis of rats treated with finasteride, a steroid-based inhibitor of 5alpha-reductase. The 5alpha-reductase is known to exist in two isoforms. Both 5alpha-red1 and 5alpha-red2 catalyse the irreversible conversion of T into DHT. Formation of DHT in the epididymis is mostly due to the action of 5alpha-red2 and finasteride is more potent inhibitor of this isoform. Rats were treated with finasteride for 56 days covering the duration of one spermatogenesis (four cycles of the seminiferous epithelium). Although E-SOD mRNA is normally expressed in cells of cauda but not of caput epididymis, treatment with finasteride produced the E-SOD transcript in cells of caput epididymis too. The GPX5 transcript was detected in cells of caput epididymis of control and experimental rats, but the level of expression measured densitometrically was significantly lower in finasteride-treated rats. The immunoexpression of Cu/ZnSOD was also changed in epididymis of finasteride-treated rats. Finasteride appears to change the pattern of expression of antioxidant enzymes and may alter the protective function of the epididymis in relation to spermatozoa.

Influence of antibiotics on growth dynamics and movement ability of Salmonella rods.

Variety of traits important in diagnostics and epidemiology of pathogenic microorganisms may change due to antibiotics. Movement ability, that is characteristic for every serovar except from Salmonella Gallinarum-Pullorum, is important to salmonellas. In own experiments using semi-fluid MSRV medium, it was found that a decrease in salmonella sensibility to selected antibiotics and chemiotherapeutics due to passage might lead to weakening of its movement ability. Movement ability of all strains (S. Enteritidis, S. Dublin, S. Typhimurium) after passage with amoxycillin, neomycin, colistin and enrofloxacin became weakened as compared to results achieved before passage. The strongest inhibition of movement ability was most often observed in strains after passage on medium with colistin. It seems to be associated with the action mechanism of the antibiotic. Colistin injuries cellular membranes, where flagella (active motoric organ of Salmonella) are anchored. Appearance of drug-resistance as a result of passage at the presence of antibiotics may cause variability of biochemical properties of Salmonella rods and leads to weakening of movement ability of ciliated Salmonella.

Morphology of the testis and the epididymis in rats with dihydrotestosterone (DHT) deficiency.

The aim of the study was to estimate morphology in the testis and epididymis of adult rats, treated with finasteride for 28 days (the time period of two seminiferous epithelium cycles) and 56 days (the time period of one spermatogenesis). A 28 days long DHT deficiency did not significantly influence the structure of seminiferous epithelium. After 56 days of treatment, finasteride induced sloughing of immature genninal cells (spermatids and rarely pachytene spermatocytes) into the lumen of the seminiferous tubules. A reduced content of spermatozoa was observed in the lumen of rat epididymis in rats with 56-day-long deficiency. The results indicated that 5alpha-reductase 2 activity is important for the maintenance of spermatogenesis. The decreased content of spermatozoa in the epididymal lumen of rats, treated with finasteride during one course of spermatogenesis, could reflect seminiferous epithelium condition.

Regulation of the insulin-like growth factor system by acute acidosis.

Many catabolic conditions are characterized by disturbances in acid-base balance and concomitant alterations in the insulin-like growth factor (IGF) system. However, the influence of acidosis per se on the various components of the IGF system has not been extensively examined. The purpose of the present study was to determine the effect of acute metabolic acidosis on the plasma and tissue concentrations of IGF-I and the various IGF-binding proteins (IGFBPs). Conscious unrestrained fasted rats were infused iv with either 0.2 N HCl or an equal volume of saline for 4 h. The arterial blood pH decreased within 60 min after starting the HCl infusion and remained lower than time-matched control values for the entire experimental protocol. Although the plasma IGF-I concentration fell gradually and was reduced by 30%, compared to time-matched control values, GH levels were unaltered. The IGF-I content of tissues collected at the conclusion of the experiment was increased in liver (35%) and kidney (63%), and unchanged in skeletal muscle. However, whereas acidosis moderately increased IGF-I messenger RNA abundance in liver, no significant alteration in IGF-I expression was detected in kidney. Acidosis also increased the plasma levels of IGFBP-1 and -2 as well as the IGFBP-1 content of liver and kidney. In contrast, the concentration of intact IGFBP-3 was decreased in acid-infused rats, and this reduction was associated with an increased rate of IGFBP-3 protease activity. Acidotic rats demonstrated unremarkable changes in the plasma concentrations of glucose and insulin, but corticosterone levels were elevated throughout the experiment. The results of the present study demonstrate that in the absence of underlying pathology, acute metabolic acidosis decreases circulating levels of IGF-I, probably by increasing renal clearance of the peptide, not by decreasing hepatic IGF-I synthesis.

Alterations in hepatic production and peripheral clearance of IGF-I after endotoxin.

Lipopolysaccharide (LPS) produces a rapid and sustained reduction in the circulating concentration of insulin-like growth factor I (IGF-I), which may be responsible, in part, for the alterations in protein metabolism observed in these animals. The purpose of the present study was to determine whether this drop was due to a decreased hepatic production of IGF-I and/or an increased clearance of the peptide from the blood. Four hours after intravenous injection of LPS the plasma IGF-I concentration was decreased 50%. IGF-I release by in situ perfused livers from control rats was constant throughout the 60-min perfusion period and averaged 111 +/- 3 ng/min. In contrast, hepatic IGF-I output was decreased 46% by in vivo LPS. In contrast, livers from LPS-injected rats released more IGF binding proteins-1, -2 and -4 than did control livers. Hepatic cell isolation indicated that LPS decreased the IGF-I content in Kupffer and parenchymal cells, but not endothelial cells, by approximately 45%. Pharmacokinetic analysis of blood 125I-IGF-I decay curves indicated that the half-life for whole body clearance of 125I-IGF-I from the circulation was not altered by LPS. However, LPS increased 125I-IGF-I uptake by spleen, liver, lung, and kidney while decreasing uptake by the pancreas and gastrointestinal tract. These results indicate that the LPS-induced decrease in blood IGF-I concentration is primarily due to a reduction in hepatic production, not a change in whole body peptide clearance, and that a decreased production by both parenchymal and Kupffer cells contributes to this alteration.

Synthesis and pharmacological investigations of 3-(aminoalkylene)-1-aryl-2-thioxo-4,5-imidazolidinedione and 2,4,5-imidazolidinetrione derivatives.

New derivatives of 2-thioxo-4,5-imidazolidinedione (3-6, 10, 11) and 2,4,5-imidazolidinetrione (7, 12) were synthesized by N,N'-acylation of asymmetric thioureas and ureas by oxalyl chloride. The obtained compounds were screened for their central action, mainly anticonvulsant activity.

1,3-disubstituted 2-thioxo-4,5-imidazolidinediones and 2,4,5-imidazolidinetriones and their anticonvulsant activity.

New unsymmetrically 1,3-disubstituted derivatives of 2-thioxo-4,5-imidazolidinedione (3, 5-7, 9-12) and 2,4,5-imidazolidinetrione (2, 8, 13-15) were synthesized by condensation of the respective thioureas and ureas with oxalyl chloride. They were screened for their central, mainly anticonvulsant activity and only compound 8 revealed antianxiety and antiepileptic properties.

Synthesis and pharmacological activity of some 2,3-dihydrofuran-2,3-dione derivatives.

New 2,3-dihydrofuran-2,3-dione derivatives (1-11) were obtained by condensation of oxalyl chloride with Schiff bases of acetyl- or benzoylacetone and aromatic amines. These compounds showed only weak sedative action and weak analgesic effect.