- Email: [email protected]
Methionine effect on DNA fragmentation during experimentally-induced cholestasis
Poster Sessions
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(50 mg/kg b. w. i. p. daily during 7 days). The animals were killed after seven days treatment. Results: Activity of gamma-glutamyl-transpeptidase (GGT) and alkaline phosphatase (AP) were significantly increased in cholestatic rats (24.15 4- 1.87 vs. control 0.93 4- 0.43 U/l; 851.2 4- 21.47 vs. control 235.75 49.91, p > 0.001). Activity of both enzymes showed significant depletion in cholestatic animals treated with methionine (63.76% for GGT and 50.47% for AP); with vitamin B 12 (56.1% GGT; 65.4% AP) and with both agents (69.59% GGT; 62.3% AP). Conclusion: Since methionine and vitamin B12 together could restore membrane fluidity in cholestatic damaged hepatocytes those agents could be important in cholestatic therapy.
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DNA ADDUCTS IN CHOLANGIOCYTES - EVIDENCE FOR CHEMICAL CARCINOGENS IN THE RISE OF BILE DUCT CANCER
Shahid Khan 1, Paul Carmichael 2, Simon Taylor-Robinson 1, Nagy Habib 3, Howard Thomas 1. 1Liver Unit, Faculty of Medicine,
Imperial College, London, England; 2Departmentof Biological Chemistry, Facultyof Medicine, Imperial College, London, England; 3Department of Surgery, Faculty of Medicine, Imperial College, London, England, UK Background: Mortality from cholangiocarcinoma (CCA) has risen steeply over the past 20 years. The cause remains unexplained. DNA adducts result from covalent carcinogen binding to nucleotides, are pro-mutagenic and demonstrate exposure to DNA-damaging agents. They are a key step in toxin-induced carcinogenesis. Hypothesis: The increase in CCA is caused by a rise in genotoxic environmental agent(s), causing cholangiocyte DNA damage. Aims: To compare CCA, tumour-adjacent and non-cancer biliary tissue, for DNA adducts as a biomarker of genotoxin exposure. Methods: DNA from 28 CCA tissues, with adjacent non-tumour tissue samples from the same patients in 24 cases; and from bile ducts from 7 noncancer patients (undergoing cholecystectomy for gallstones) were investigated for the presence of DNA adducts using 32P-postlabelling. Relative adduct labelling values (RAL, adducts/108 nucleotides) were quantified. Results: No difference was found in RALs between DNA from CCA tissue (mean 14, range 1-48) and tumour-adjacent tissue DNA (mean 14, range 1-52). RALs were significantly higher in tissue from CCA patients than from non-cancer patients (mean 6, range 1-31, p = 0.04, Mann-Whitney test). Different adduct patterns were also seen in CCA compared to noncancer patients. Conclusion: Quantitative and qualitative differences in adducts between cancer and non-cancer patients support the hypothesis that genotoxins play a role in the development of CCA.
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METHIONINE EFFECT ON DNA FRAGMENTATION DURING EXPERIMENTALLY-INDUCED CHOLESTASIS
G. Kocic, G. Bjelakovic, D. Sokolovic, T. Jevtovic, D. Pavlovic, G.B. Bjelakovic, R. Kocic. Institute of Biochemistry and Clinical Center
Faculty of Medicine, Nis, Yugoslavia Introduction and Aim: Internucleosomal cleavage of DNA has been regarded as the biochemical halmark of apoptosis, a pattern of cell death which occurs in cholestasis, but the informations are lacking over which endonuclease is reponsible for DNA digestion. Since methionine-an endogenous methyl group donor is an interesting new therapeutic agent in several hepatic diesease conditions, including cholestasis, the aim was also to evaluate the effect of methionine on nuclear DNA fragmentation during cholestasis. Experiment: The common bile duct was ligated (CBDL) and rats were randomised to either an untreated group or to treatment with methionine CBDL+M (50 mg/24 h), and intact rats were also randomised to either
an untreated-control group or treated with methionine (50 mg/24 h), for a period of 7 days. One group underwent only sham laparatomy. Results and Discussion: Bilirnbine level (Ixmogl) was not significantly different between CBDL group (270 4- 22.43) and CBDL+M (260 412.00), but significantly (p < 0.001) vs control (0.5 4- 0.01) and M (0.3 4- 0.02). The apoptotic effect of cholestasis is probably triggered by the activation of of Mg 2÷ -dependent DN-ase I (3.27 4- 0.69 vs control 1.08 4- 0.36 U/g prot. vs sham-operated 1.11 4- 0.16 p < 0.001), explained by the significant increase of intarcellular Mg 2÷ that occurs in cholestasis, and by the direct caspase3-induced proteolytic enzyme activation. Our data suggest for the first time that the increase of acidic DNase II (2.73 40.91 vs control 1.68 4- 0.45 vs sham-operated 1.78 4- 0.23 U/g prot. p < 0.001), as a result of activation (associated with caspase-dependent proteolysis and cellular acidification) and nuclear translocation, may participate in the death program of cellular events culminating in cholestasis-induced apoptosis. Beside of the mandatory role of methionine in membrane stability, the reverting effect on terminal apoptotic reaction could be partial, because of the decrease in DNase I activity (2.52 4- 0.30 p < 0.05), but not in DNase II (2.82 4- 0.57), while given to intact rats did not alter DNase I significantly (1.01 ± 0.42), but decreased DNase II (1.53 4- 0.09 p < 0.05). Conclusion: Since it was documented that both of the investigated enzymes can be included in DNA cleavage site, benefitial effect of methionine may be only partial on DNA fragmentation during cholestasis.
•
GLIBENCLAMIDE INDUCES DUCTAL SECRETION STIMULATING VESICULAR TRANSPORT IN CHOLANGIOCYTES
Carlo Spirlil, Romina Fiorotto 1, Elena Duner 1, Sara Masier 1, Massimiliano Cadamuro 1,2, Mario Strazzabosco 1.2. lDept of Medical and
Surgical Sciences, University of Padova; 2Division of Gastroenterology, Ospedali Riuniti di Bergamo, Italy Glibenclamide (GL), stimulates bile flow in biliary epithelium via Ca2+ and cAMP independent mechanisms. GL effects are retained in cholestatic rats, making this class of compounds an attractive tool to stimulate fluid secretion in biliary tract diseases. Our aim was to indagate the molecular mechanisms involved in the choleretic effect of GL. GL and its metabolites were present, by HPLC, in micromolar amounts in bile collected from isolated rat liver perfused with 100 mM GL, but not in the overnatant of normal rat cholangiocyte monolayers (NRC-1) exposed to 100 mM GL on the basolateral side, indicating that choleresis in the isolated rat bile duct units (IBDU) is not due to GL transport by cholangiocytes. GL-induced secretion (video-optical planimetry) was inhibited by C1- channel blockers, but not by omission of HCO3 from the perfusion media suggesting its dependence on C l - channels, but not on C 1 - / H C O 3 - exchanger. By RT/PCR the sulfonylurea receptor SUR-2B mRNA was expressed in cholangiocytes. GL effects were mimicked by tolbutamide and inhibited by diazoxide. GLstimulated secretion in IBDU was inhibited by p-glycoproteins inhibitors (verapamil and GF120918) and by tamoxifene, an inhibitor of a mdrllike protein, that in pancreatic beta-cells, couples SUR to granular C l channels (PNAS 96: 5539, 1999). Finally, GL choleresis was inhibited by worthmannin, LY294002, colchicine, but not by lumicolchicine. These findings suggest that, sulfonylureas interacting with the SUR receptor may promote exocytosis in cholangiocytes by a mechanism involving mdr-like proteins and C1- transport.
142
(50 mg/kg b. w. i. p. daily during 7 days). The animals were killed after seven days treatment. Results: Activity of gamma-glutamyl-transpeptidase (GGT) and alkaline phosphatase (AP) were significantly increased in cholestatic rats (24.15 4- 1.87 vs. control 0.93 4- 0.43 U/l; 851.2 4- 21.47 vs. control 235.75 49.91, p > 0.001). Activity of both enzymes showed significant depletion in cholestatic animals treated with methionine (63.76% for GGT and 50.47% for AP); with vitamin B 12 (56.1% GGT; 65.4% AP) and with both agents (69.59% GGT; 62.3% AP). Conclusion: Since methionine and vitamin B12 together could restore membrane fluidity in cholestatic damaged hepatocytes those agents could be important in cholestatic therapy.
~-~
DNA ADDUCTS IN CHOLANGIOCYTES - EVIDENCE FOR CHEMICAL CARCINOGENS IN THE RISE OF BILE DUCT CANCER
Shahid Khan 1, Paul Carmichael 2, Simon Taylor-Robinson 1, Nagy Habib 3, Howard Thomas 1. 1Liver Unit, Faculty of Medicine,
Imperial College, London, England; 2Departmentof Biological Chemistry, Facultyof Medicine, Imperial College, London, England; 3Department of Surgery, Faculty of Medicine, Imperial College, London, England, UK Background: Mortality from cholangiocarcinoma (CCA) has risen steeply over the past 20 years. The cause remains unexplained. DNA adducts result from covalent carcinogen binding to nucleotides, are pro-mutagenic and demonstrate exposure to DNA-damaging agents. They are a key step in toxin-induced carcinogenesis. Hypothesis: The increase in CCA is caused by a rise in genotoxic environmental agent(s), causing cholangiocyte DNA damage. Aims: To compare CCA, tumour-adjacent and non-cancer biliary tissue, for DNA adducts as a biomarker of genotoxin exposure. Methods: DNA from 28 CCA tissues, with adjacent non-tumour tissue samples from the same patients in 24 cases; and from bile ducts from 7 noncancer patients (undergoing cholecystectomy for gallstones) were investigated for the presence of DNA adducts using 32P-postlabelling. Relative adduct labelling values (RAL, adducts/108 nucleotides) were quantified. Results: No difference was found in RALs between DNA from CCA tissue (mean 14, range 1-48) and tumour-adjacent tissue DNA (mean 14, range 1-52). RALs were significantly higher in tissue from CCA patients than from non-cancer patients (mean 6, range 1-31, p = 0.04, Mann-Whitney test). Different adduct patterns were also seen in CCA compared to noncancer patients. Conclusion: Quantitative and qualitative differences in adducts between cancer and non-cancer patients support the hypothesis that genotoxins play a role in the development of CCA.
[-~
METHIONINE EFFECT ON DNA FRAGMENTATION DURING EXPERIMENTALLY-INDUCED CHOLESTASIS
G. Kocic, G. Bjelakovic, D. Sokolovic, T. Jevtovic, D. Pavlovic, G.B. Bjelakovic, R. Kocic. Institute of Biochemistry and Clinical Center
Faculty of Medicine, Nis, Yugoslavia Introduction and Aim: Internucleosomal cleavage of DNA has been regarded as the biochemical halmark of apoptosis, a pattern of cell death which occurs in cholestasis, but the informations are lacking over which endonuclease is reponsible for DNA digestion. Since methionine-an endogenous methyl group donor is an interesting new therapeutic agent in several hepatic diesease conditions, including cholestasis, the aim was also to evaluate the effect of methionine on nuclear DNA fragmentation during cholestasis. Experiment: The common bile duct was ligated (CBDL) and rats were randomised to either an untreated group or to treatment with methionine CBDL+M (50 mg/24 h), and intact rats were also randomised to either
an untreated-control group or treated with methionine (50 mg/24 h), for a period of 7 days. One group underwent only sham laparatomy. Results and Discussion: Bilirnbine level (Ixmogl) was not significantly different between CBDL group (270 4- 22.43) and CBDL+M (260 412.00), but significantly (p < 0.001) vs control (0.5 4- 0.01) and M (0.3 4- 0.02). The apoptotic effect of cholestasis is probably triggered by the activation of of Mg 2÷ -dependent DN-ase I (3.27 4- 0.69 vs control 1.08 4- 0.36 U/g prot. vs sham-operated 1.11 4- 0.16 p < 0.001), explained by the significant increase of intarcellular Mg 2÷ that occurs in cholestasis, and by the direct caspase3-induced proteolytic enzyme activation. Our data suggest for the first time that the increase of acidic DNase II (2.73 40.91 vs control 1.68 4- 0.45 vs sham-operated 1.78 4- 0.23 U/g prot. p < 0.001), as a result of activation (associated with caspase-dependent proteolysis and cellular acidification) and nuclear translocation, may participate in the death program of cellular events culminating in cholestasis-induced apoptosis. Beside of the mandatory role of methionine in membrane stability, the reverting effect on terminal apoptotic reaction could be partial, because of the decrease in DNase I activity (2.52 4- 0.30 p < 0.05), but not in DNase II (2.82 4- 0.57), while given to intact rats did not alter DNase I significantly (1.01 ± 0.42), but decreased DNase II (1.53 4- 0.09 p < 0.05). Conclusion: Since it was documented that both of the investigated enzymes can be included in DNA cleavage site, benefitial effect of methionine may be only partial on DNA fragmentation during cholestasis.
•
GLIBENCLAMIDE INDUCES DUCTAL SECRETION STIMULATING VESICULAR TRANSPORT IN CHOLANGIOCYTES
Carlo Spirlil, Romina Fiorotto 1, Elena Duner 1, Sara Masier 1, Massimiliano Cadamuro 1,2, Mario Strazzabosco 1.2. lDept of Medical and
Surgical Sciences, University of Padova; 2Division of Gastroenterology, Ospedali Riuniti di Bergamo, Italy Glibenclamide (GL), stimulates bile flow in biliary epithelium via Ca2+ and cAMP independent mechanisms. GL effects are retained in cholestatic rats, making this class of compounds an attractive tool to stimulate fluid secretion in biliary tract diseases. Our aim was to indagate the molecular mechanisms involved in the choleretic effect of GL. GL and its metabolites were present, by HPLC, in micromolar amounts in bile collected from isolated rat liver perfused with 100 mM GL, but not in the overnatant of normal rat cholangiocyte monolayers (NRC-1) exposed to 100 mM GL on the basolateral side, indicating that choleresis in the isolated rat bile duct units (IBDU) is not due to GL transport by cholangiocytes. GL-induced secretion (video-optical planimetry) was inhibited by C1- channel blockers, but not by omission of HCO3 from the perfusion media suggesting its dependence on C l - channels, but not on C 1 - / H C O 3 - exchanger. By RT/PCR the sulfonylurea receptor SUR-2B mRNA was expressed in cholangiocytes. GL effects were mimicked by tolbutamide and inhibited by diazoxide. GLstimulated secretion in IBDU was inhibited by p-glycoproteins inhibitors (verapamil and GF120918) and by tamoxifene, an inhibitor of a mdrllike protein, that in pancreatic beta-cells, couples SUR to granular C l channels (PNAS 96: 5539, 1999). Finally, GL choleresis was inhibited by worthmannin, LY294002, colchicine, but not by lumicolchicine. These findings suggest that, sulfonylureas interacting with the SUR receptor may promote exocytosis in cholangiocytes by a mechanism involving mdr-like proteins and C1- transport.