Indukce hemoxygenasy a biologická úloha jejích metabolických produktů.

Hemoxygenasa (HMOX) je enzym katalyzující první a rychlost limitující reakci štěpení hemu. Jejím působením vzniká oxid uhelnatý (CO), železnatý ion a biliverdin, který je následně redukován na bilirubin. CO byl před objevem mechanismu reakce katalyzované HMOX a ještě dlouho poté považován pouze za toxický produkt bez pozitivního významu pro lidský organismus. Podobně byl vnímán i bilirubin, marker jaterního poškození. Výsledky studií z posledních let ovšem ukazují, že HMOX i její metabolické produkty hrají významnou roli v řadě fyziologických procesů stejně tak jako i v obraně před procesy patologickými. Cílem této práce bylo objasnit roli HMOX a jejích metabolicky aktivních produktů, především CO a bilirubinu, in vivo a in vitro. Zabývali jsme se studiem účinků CO, kdy jsme jako první popsali tkáňovou distribuci a farmakokinetiku inhalovaného CO u potkanů. Na modelu systémové sepse vyvolané endotoxinem u potkanů jsme zjistili, že inhalace CO je spojena s protizánětlivými a hepatoprotektivními účinky. U modelu cholestázy indukované ethinylestradiolem jsme prokázali anticholestatické účinky HMOX. Indukce HMOX1 jejím substrátem hemem zvyšovala expresi jaterních transportérů a tím podpořila tok žluče u cholestatických potkanů, zároveň usnadnila efektivní clearance konjugovaných žlučových kyselin...Heme oxygenase (HMOX) catalyzes first and rate-limiting step in heme degradation. By its action, carbon monoxide (CO), ferrous iron and biliverdin which is subsequently reduced to bilirubin are produced. Before discovery of HMOX reaction mechanism, CO was considered only a toxic waste product without any significant importance for human organism. Bilirubin, marker of liver dysfunction, has been also exposed to similar perception. But results from past decades show that HMOX and its metabolic products play an important role in number of physiological as well as defense against pathophysiological processes. The aim of this thesis was to clarify the role of HMOX and its metabolic products, presumably CO and bilirubin, in vivo and in vitro. We focused on the role of CO in a rat model of lipopolysaccharide-induced cholestasis. We were first to describe tissue distribution and pharmacokinetics of inhaled CO in this animal model and found out that CO inhalation is associated with anti-inflammatory and hepatoprotective effects. In a rat model of ethinylestradiol-induced cholestasis, we demonstrated the anticholestatic effect of HMOX. The induction of HMOX by its substrate heme increased the expression of liver transporters thereby increasing bile flow and simultaneously facilitated effective clearance of...Institute of Medical Biochemistry and Laboratory Medicine First Faculty of MedicineÚstav lékařské biochemie a laboratorní diagnostiky 1. LF UK a VFN1. lékařská fakultaFirst Faculty of Medicin

Heme oxygenase-1 may affect cell signalling via modulation of ganglioside composition

Heme oxygenase 1 (Hmox1), a ubiquitous enzyme degrading heme to carbon monoxide, iron, and biliverdin, is one of the cytoprotective enzymes induced in response to a variety of stimuli, including cellular oxidative stress. Gangliosides, sialic acid-containing glycosphingolipids expressed in all cells, are involved in cell recognition, signalling, and membrane stabilization. Their expression is often altered under many pathological and physiological conditions including cell death, proliferation, and differentiation. The aim of this study was to assess the possible role of Hmox1 in ganglioside metabolism in relation to oxidative stress. The content of liver and brain gangliosides, their cellular distribution, and mRNA as well as protein expression of key glycosyltransferases were determined in Hmox1 knockout mice as well as their wild-type littermates. To elucidate the possible underlying mechanisms between Hmox1 and ganglioside metabolism, hepatoblastoma HepG2 and neuroblastoma SH-SY5Y cell lines were used for in vitro experiments. Mice lacking Hmox1 exhibited a significant increase in concentrations of liver and brain gangliosides and in mRNA expression of the key enzymes of ganglioside metabolism. A marked shift of GM1 ganglioside from the subsinusoidal part of the intracellular compartment into sinusoidal membranes of hepatocytes was shown in Hmox1 knockout mice. Induction of oxidative stress by chenodeoxycholic acid in vitro resulted in a significant increase in GM3, GM2, and GD1a gangliosides in SH-SY5Y cells and GM3 and GM2 in the HepG2 cell line. These changes were abolished with administration of bilirubin, a potent antioxidant agent. These observations were closely related to oxidative stress-mediated changes in sialyltransferase expression regulated at least partially through the protein kinase C pathway. We conclude that oxidative stress is an important factor modulating synthesis and distribution of gangliosides in vivo and in vitro which might affect ganglioside signalling in higher organisms

Induction of heme oxygenase and biological role of its metabolic products.

Heme oxygenase (HMOX) catalyzes first and rate-limiting step in heme degradation. By its action, carbon monoxide (CO), ferrous iron and biliverdin which is subsequently reduced to bilirubin are produced. Before discovery of HMOX reaction mechanism, CO was considered only a toxic waste product without any significant importance for human organism. Bilirubin, marker of liver dysfunction, has been also exposed to similar perception. But results from past decades show that HMOX and its metabolic products play an important role in number of physiological as well as defense against pathophysiological processes. The aim of this thesis was to clarify the role of HMOX and its metabolic products, presumably CO and bilirubin, in vivo and in vitro. We focused on the role of CO in a rat model of lipopolysaccharide-induced cholestasis. We were first to describe tissue distribution and pharmacokinetics of inhaled CO in this animal model and found out that CO inhalation is associated with anti-inflammatory and hepatoprotective effects. In a rat model of ethinylestradiol-induced cholestasis, we demonstrated the anticholestatic effect of HMOX. The induction of HMOX by its substrate heme increased the expression of liver transporters thereby increasing bile flow and simultaneously facilitated effective clearance of..

Characterisation of iodo- plus mesosulfuron resistance in an Alopecurus myosuroides Huds. Population from the Czech Republic

The intensification of Alopecurus myosuroides Huds. (black-grass) is becoming a major problem due to its growing resistance to a broad spectrum of acetolactate synthase (ALS)-inhibiting herbicides. Hence, the present study was conducted to evaluate the resistance level of a black-grass population to iodosulfuron plus mesosulfuron and to identify the underlying resistance mechanism. Dose-response studies revealed that the resistance population is 22 times less sensitive to iodosulfuron plus mesosulfuron than the susceptible population. The probable resistance mechanism identified was the target-site substitution of proline (Pro) by threonine (Thr) at the 197th position of the ALS enzyme. Furthermore, whole plant response bioassay experiments demonstrated that this population is also resistant to pinoxaden, chlorotoluron, diflufenican plus pendimethalin plus chlorotoluron, fenoxaprop and flufenacet plus diflufenican. In summary, the current findings recommend using alternative herbicides in integrated weed management to interrupt the possible evolution of herbicide resistance in these species

Isolated Silymarin Flavonoids Increase Systemic and Hepatic Bilirubin Concentrations and Lower Lipoperoxidation in Mice

Bilirubin is considered to be one of the most potent endogenous antioxidants in humans. Its serum concentrations are predominantly affected by the activity of hepatic bilirubin UDP-glucuronosyl transferase (UGT1A1). Our objective was to analyze the potential bilirubin-modulating effects of natural polyphenols from milk thistle (Silybum marianum), a hepatoprotective herb. Human hepatoblastoma HepG2 cells were exposed to major polyphenolic compounds isolated from milk thistle. Based on in vitro studies, 2,3-dehydrosilybins A and B were selected as the most efficient compounds and applied either intraperitoneally or orally for seven days to C57BL/6 mice. After, UGT1A1 mRNA expression, serum, intrahepatic bilirubin concentrations, and lipoperoxidation in the liver tissue were analyzed. All natural polyphenols used increased intracellular concentration of bilirubin in HepG2 cells to a similar extent as atazanavir, a known bilirubinemia-enhancing agent. Intraperitoneal application of 2,3-dehydrosilybins A and B (the most efficient flavonoids from in vitro studies) to mice (50 mg/kg) led to a significant downregulation of UGT1A1 mRNA expression (46±3% of controls, p<0.005) in the liver and also to a significant increase of the intracellular bilirubin concentration (0.98±0.03vs.1.21±0.02 nmol/mg, p<0.05). Simultaneously, a significant decrease of lipoperoxidation (61±2% of controls, p<0.005) was detected in the liver tissue of treated animals, and similar results were also observed after oral treatment. Importantly, both application routes also led to a significant elevation of serum bilirubin concentrations (125±3% and 160±22% of the controls after intraperitoneal and oral administration, respectively, p<0.005 in both cases). In conclusion, polyphenolic compounds contained in silymarin, in particular 2,3-dehydrosilybins A and B, affect hepatic and serum bilirubin concentrations, as well as lipoperoxidation in the liver. This phenomenon might contribute to the hepatoprotective effects of silymarin

Antiproliferative effects of carbon monoxide on pancreatic cancer

Background - Carbon monoxide, the gaseous product of heme oxygenase, is a signalling molecule with a broad spectrum of biological activities. The aim of this study was to investigate the effects of carbon monoxide on proliferation of human pancreatic cancer. Methods - In vitro studies were performed on human pancreatic cancer cells (CAPAN-2, BxPc3, and PaTu-8902) treated with a carbon monoxide-releasing molecule or its inactive counterpart, or exposed to carbon monoxide gas (500 ppm/24 h). For in vivo studies, pancreatic cancer cells (CAPAN-2/PaTu-8902) were xenotransplanted subcutaneously into athymic mice, subsequently treated with carbon monoxide-releasing molecule (35 mg/kg b.w. i.p./day), or exposed to safe doses of carbon monoxide (500 ppm 1 h/day; n = 6 in each group). Results - Both carbon monoxide-releasing molecule and carbon monoxide exposure significantly inhibited proliferation of human pancreatic cancer cells (p < 0.05). A substantial decrease in Akt phosphorylation was observed in carbon monoxide-releasing molecule compared with inactive carbon monoxide-releasing molecule treated cancer cells (by 30–50%, p < 0.05). Simultaneously, carbon monoxide-releasing molecule and carbon monoxide exposure inhibited tumour proliferation and microvascular density of xenotransplanted tumours (p < 0.01), and doubled the survival rates (p < 0.005). Exposure of mice to carbon monoxide led to an almost 3-fold increase in carbon monoxide content in tumour tissues (p = 0.006). Conclusion - These data suggest a new biological function for carbon monoxide in carcinogenesis, and point to the potential chemotherapeutic/chemoadjuvant use of carbon monoxide in pancreatic cancer