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The isoflavone genistein (GEN) undergoes an enterohepatic circulation
I-IEPATOLOGY Vol. 22, No. 4, P t . 2, 1995
AASLD
5 6 ] - LIVER AND INTESTINAL PROPOXYPHENE METABOLISMS : AN EXAMPLE OF DISCREPRANCY IN DRUG PRE-SYSTEMIC BIOTRANSFORMATION IN THE RAT. Y. Horsmans. A. Saliez. V. Van Den Berne. J.P. Desaoer. A.P. Geubel. S. Pauwels and L. Lambotte. Dept. of Gastroenterology] Experimental Surgery and Pharmaeotherapy Laboratories, Catholic University of Louvain, Brussels.
ABSTRACTS
562
THE ISOFLAVONE GENISTEIN (GEN) UNDERGOES AN ENTEROHEPATIC CIRCULATION. J. Sfakianos. S. Barnes. Dept. Pharrnaeol., and Comprehensive Cancer Center Mass Spectrometry SharedFacility, University of Alabama at Birmingham, AL 35294. It has been claimed that GEN, a component in soy responsible for a reduction in cancer and cardiovascular disease risk, has a poor bioavailability based on its low urinary excretion (J. Nutr.124, 825-832, 1994). However, no direct experiments on the intestinal absorption of GEN have been reported. In the present study, t4C-labeled GEN was administered to anesthetized, adult female rats fitted with an indwelling biliary catheter. Biles were collected in 20 minute intervals for up to 4 hours. Metabolites of GEN were separated by reversed-phase HPLC and identified by on-line electrospray ionization-mass spectrometry (ESIMS). Results: Perfusion of t4C-GEN through a 10 cm closed loop of proximal small intestine led to the rapid appearance of 14C-radioactivity in bile, the % recovery being independent of the dose over the range 1.757.0 Ilg/min. Following infusions of GEN for 1 h into the proximal small intestine, 65-70% of the dose was recovered in bile over 4 h, the peak of radioactivity appearing after 1 h. HPLC-ESI-MS revealed that GEN was excreted in bile as its glueuronide (GEN-GIcA). Less than 2% of the dose was excreted in the urine, principally as GEN-GlcA. When t4C-GENGlcA recovered from bile was reinfused duodenally, 25-30% of the dose was recovered in the bile over a 4 hour period. The unabsorbed 14Cradioactivity which remained in the small intestine was mostly GEN. When GEN-GlcA was infused into the mid-small intestine, biliary recovery, over 4 h, of tnC-radioactivity was increased to 65-70%, again as GEN-GlcA. Urinary excretion of 14C-radioactivity during this period was 6% of dose. Conclusion: In rats, GEN and its metabolite GENGlcA are both well absorbed from the small intestine and undergo an enterohepatic circulation. GEN-GlcA is hydrolyzed to GEN before intestinal absorption. The low levels of GEN that appear in the urine while consuming a soy diet, therefore, are not a consequence of its poor intestinal absorption, but rather a combination of confinement in an enterohepatic circulation by intestinal absorption and hepatobiliary transport into bile and possibly selective renal transport/excretion mechanisms between GEN and other isoflavones.
Paradoxical Effects of Tamoxifen in Liver. P.K. Eaton and M.J. Epl¢y. VA Medical Center, Pittsburgh PA 15240 and University of Pittsburgh School of Medicine, Pittsburgh PA 15261
Introduction: Tamoxifen (Tam) is widely used as an amiestrogen in the treatment of breast cancer, and has been used in some recent clinical trials as a therapeutic agent in hepatocellular cancer (HCC). Because the liver is a key organ in homeostasis of endogenous sex hormones and metabolism of Tam, the effect of Tam treatment on several parameters of liver function was of interest. Methods: Male and female adult rats were treated for 7 days with Tam or estradiol (E2); the effects of these agents on hepatic steroid metabolism and on the hepatic expression of an estrogenresponsive protein ceruloplasmin (CP) were assessed. Results: In both sexes, Tam treatment resulted in significant stimulation of CP levels. In males, Tam resulted in a 150% increase in CP, as compared to a 120% induced by E2 treatment. In females, Tam increased CP by 150%, as compared to a 155% induction with E2. These increases in CP by Tam treatment are indicative of a strong estrogenic response by the liver of these rats. However, there were profound differences between Tam and E2 in their effect on estrogen metabolism. Whereas E2 treatment caused a significant loss in activity of two male-specific isozymes of key estrogen metabolizing enzymes (estrogen sulfotransferase, a 90 % loss, and estrogen 2-hydroxylase, a 50% loss), Tam treatment caused only slight and not significant reductions in these activities. Tam did not affect serum testosterone levels in the males, whereas E2 treatment resulted in a 90% suppression. In females, Tam had no effect on female-specific estrogen 2hydroxylation, whereas E~ treatment stimulated this enzyme slightly. Conclusions: Tam treatment results in an estrogenic stimulation of liver in both sexes in that serum CP is enhanced by this agent, and thus was similar to E2 treatment. However, unlike E2, Tam treatment does not suppress female- or male-specific estrogen metabolism nor testosterone levels in male rats. Thus, Tam does not alter sex-specific metabolism of endogenous estrogens, yet produces a clear estrogenic response in liver.
Presystemie metabolism responsible for the reduction of the systemic concentration of a drug given by oral administration is attributed mainly to the liver with some minor intestinal participation. Both are generally considered as a whole and supposed to differ only quantitatively. The present study of the demethylation of the widely prescribed analgesic propoxyphene (DP) suggests that the metabolism greatly differs within these two organs. A tracer dose of (14C-)DP (Amersham, U.K.) was administered either in the femoral vein (IV) or centrally in the left cardiac ventdcule (IC) or in the gastroduodenal vein (IP) of male Wistar rats. Mean (± S.D.) CO2 excretion, during the first 2 hours was 20.1 (± 3.8)% of the administered dose after IP (n=6) and only 11.8 (5- 1.3)% (n=4) and 10.2% (n=2) after IV and IC, respectively. The same difference persisted in the 24 hours collection indicating that this latter was not a simple delay due to a larger volume of distribution. The greatest output after IP was not due to the saturation of a parallel hepatic metabolism due to a greater DP concentration since only a tracer dose was used and also since the difference persisted when DP was given in 20 minutes (n=2). DP demethyladon seems limited to the liver as the production of CO2 was reduced to 1.7% after IV administration in portoeaval shunted hepatectomized rats (n---4).It is therefore proposed that it is the amount of DP entering the liver which determines the production of CO2 and that a large part of DP is transformed extrahepatically into metabolites not subjected to further demethylation. This transformation takes place in the gut since the CO2 production was equivalent to that obtained after IV (11.5 5. 2.6%; n=6). In vitro microsomes of hepatic but not of intestinal origin generated demethylation to form a quantifiable amount of norpmpoxyphene. In conclusion, this study suggests that the prchepatic transformation of a drug may influence not only quantitatively but also qualitatively the production of its metabolites.
563
247A
564
G L U T A T H I O N E AS AN I M P O R T A N T ANTIOXIDANT AGAINST HYDROC, EN P E R O X I D E - M E D I A T E D DECRAESE IN MITOCHONDRIAL ENERGIZATION IN RAT HEPATOCYTES TREATED WITH ETHANOL. H Higuchi, I Kurose, N Watanabe, M Takaishi, S Kato, S Miura, and H lshii. Depl. qf Internal Med., Keio
lhJiv. Schmd ~!lMed. fbkyo 160. JAPAN. Therc has hccn much controvcrsy oil the clhanol induced impairment of oxygcn utili/ation. The present study was designed to evaluate the mechanism bx which cthanol modulates mitochondrial function in hepatocytes. Fo Ihat cfl'cct, hepatocytes were isolated from male Wistar rats, and cultured. After loading with either rhodamine 123 (Rh123), a mitochondrial membrane potential-sensitive fluorescence probe, or dichlorofluorescin 0)CF), a hydrogen peroxide-sensitive fluorescence probe, tat hepatocytcs were incubated with ethanol (50 raM). Alterations of Rhl23- and DCF-associated fluorescences in each hepatocyte were visualized under a laser scanning confocal microscope. The Rh123 fluorescence decreased to 40% and DCF fluorescence increased to 200% of the baseline valuc in each hepatocyte 30 minutes after incubation with ethanol. The decrease in Rh123 fluorescence and increase in DCF fluorescence observed after incubation with ethanol were significantly attenuated by addition of either N,N'-dimethylthiourea (10 raM), a cell membrane permeable hydrogen peroxide scavenger, or 4-methylpyrazole (500 l.tM). In contrast, 3-amino-l,2,4-triazole (1 mM), a catalase inhibitor, and maleic acid diethyl ester (MADE, 500 pM), a glutathione depictor, enhanced the ethanol-induced reduction of Rh123 fluorescence and elevation of I)CF fluorescence in hepatocytes. MADE was morepotent than ATZ to enhance these ethanol-induced fluorographic alterations. In some experiments, monochlorhimane (MCLB, 20 ~tM), a glutathionc-scnsitivc fluorescence probe; was added to the culture medium after each Rh123 study. This 2-color fluorescence study revealed that there is an inverse linear correlation between the glutathione content and the degree of ethanol-induced decrease in Rh123 fluorescence m an individual hepatocytes. Thus, the present study suggests that imracellular glutathione is an important cytoprotective factor against hydrogen peroxide-induced reduction of mitochondrial energization in ethanol-treated hepatocytes.
AASLD
5 6 ] - LIVER AND INTESTINAL PROPOXYPHENE METABOLISMS : AN EXAMPLE OF DISCREPRANCY IN DRUG PRE-SYSTEMIC BIOTRANSFORMATION IN THE RAT. Y. Horsmans. A. Saliez. V. Van Den Berne. J.P. Desaoer. A.P. Geubel. S. Pauwels and L. Lambotte. Dept. of Gastroenterology] Experimental Surgery and Pharmaeotherapy Laboratories, Catholic University of Louvain, Brussels.
ABSTRACTS
562
THE ISOFLAVONE GENISTEIN (GEN) UNDERGOES AN ENTEROHEPATIC CIRCULATION. J. Sfakianos. S. Barnes. Dept. Pharrnaeol., and Comprehensive Cancer Center Mass Spectrometry SharedFacility, University of Alabama at Birmingham, AL 35294. It has been claimed that GEN, a component in soy responsible for a reduction in cancer and cardiovascular disease risk, has a poor bioavailability based on its low urinary excretion (J. Nutr.124, 825-832, 1994). However, no direct experiments on the intestinal absorption of GEN have been reported. In the present study, t4C-labeled GEN was administered to anesthetized, adult female rats fitted with an indwelling biliary catheter. Biles were collected in 20 minute intervals for up to 4 hours. Metabolites of GEN were separated by reversed-phase HPLC and identified by on-line electrospray ionization-mass spectrometry (ESIMS). Results: Perfusion of t4C-GEN through a 10 cm closed loop of proximal small intestine led to the rapid appearance of 14C-radioactivity in bile, the % recovery being independent of the dose over the range 1.757.0 Ilg/min. Following infusions of GEN for 1 h into the proximal small intestine, 65-70% of the dose was recovered in bile over 4 h, the peak of radioactivity appearing after 1 h. HPLC-ESI-MS revealed that GEN was excreted in bile as its glueuronide (GEN-GIcA). Less than 2% of the dose was excreted in the urine, principally as GEN-GlcA. When t4C-GENGlcA recovered from bile was reinfused duodenally, 25-30% of the dose was recovered in the bile over a 4 hour period. The unabsorbed 14Cradioactivity which remained in the small intestine was mostly GEN. When GEN-GlcA was infused into the mid-small intestine, biliary recovery, over 4 h, of tnC-radioactivity was increased to 65-70%, again as GEN-GlcA. Urinary excretion of 14C-radioactivity during this period was 6% of dose. Conclusion: In rats, GEN and its metabolite GENGlcA are both well absorbed from the small intestine and undergo an enterohepatic circulation. GEN-GlcA is hydrolyzed to GEN before intestinal absorption. The low levels of GEN that appear in the urine while consuming a soy diet, therefore, are not a consequence of its poor intestinal absorption, but rather a combination of confinement in an enterohepatic circulation by intestinal absorption and hepatobiliary transport into bile and possibly selective renal transport/excretion mechanisms between GEN and other isoflavones.
Paradoxical Effects of Tamoxifen in Liver. P.K. Eaton and M.J. Epl¢y. VA Medical Center, Pittsburgh PA 15240 and University of Pittsburgh School of Medicine, Pittsburgh PA 15261
Introduction: Tamoxifen (Tam) is widely used as an amiestrogen in the treatment of breast cancer, and has been used in some recent clinical trials as a therapeutic agent in hepatocellular cancer (HCC). Because the liver is a key organ in homeostasis of endogenous sex hormones and metabolism of Tam, the effect of Tam treatment on several parameters of liver function was of interest. Methods: Male and female adult rats were treated for 7 days with Tam or estradiol (E2); the effects of these agents on hepatic steroid metabolism and on the hepatic expression of an estrogenresponsive protein ceruloplasmin (CP) were assessed. Results: In both sexes, Tam treatment resulted in significant stimulation of CP levels. In males, Tam resulted in a 150% increase in CP, as compared to a 120% induced by E2 treatment. In females, Tam increased CP by 150%, as compared to a 155% induction with E2. These increases in CP by Tam treatment are indicative of a strong estrogenic response by the liver of these rats. However, there were profound differences between Tam and E2 in their effect on estrogen metabolism. Whereas E2 treatment caused a significant loss in activity of two male-specific isozymes of key estrogen metabolizing enzymes (estrogen sulfotransferase, a 90 % loss, and estrogen 2-hydroxylase, a 50% loss), Tam treatment caused only slight and not significant reductions in these activities. Tam did not affect serum testosterone levels in the males, whereas E2 treatment resulted in a 90% suppression. In females, Tam had no effect on female-specific estrogen 2hydroxylation, whereas E~ treatment stimulated this enzyme slightly. Conclusions: Tam treatment results in an estrogenic stimulation of liver in both sexes in that serum CP is enhanced by this agent, and thus was similar to E2 treatment. However, unlike E2, Tam treatment does not suppress female- or male-specific estrogen metabolism nor testosterone levels in male rats. Thus, Tam does not alter sex-specific metabolism of endogenous estrogens, yet produces a clear estrogenic response in liver.
Presystemie metabolism responsible for the reduction of the systemic concentration of a drug given by oral administration is attributed mainly to the liver with some minor intestinal participation. Both are generally considered as a whole and supposed to differ only quantitatively. The present study of the demethylation of the widely prescribed analgesic propoxyphene (DP) suggests that the metabolism greatly differs within these two organs. A tracer dose of (14C-)DP (Amersham, U.K.) was administered either in the femoral vein (IV) or centrally in the left cardiac ventdcule (IC) or in the gastroduodenal vein (IP) of male Wistar rats. Mean (± S.D.) CO2 excretion, during the first 2 hours was 20.1 (± 3.8)% of the administered dose after IP (n=6) and only 11.8 (5- 1.3)% (n=4) and 10.2% (n=2) after IV and IC, respectively. The same difference persisted in the 24 hours collection indicating that this latter was not a simple delay due to a larger volume of distribution. The greatest output after IP was not due to the saturation of a parallel hepatic metabolism due to a greater DP concentration since only a tracer dose was used and also since the difference persisted when DP was given in 20 minutes (n=2). DP demethyladon seems limited to the liver as the production of CO2 was reduced to 1.7% after IV administration in portoeaval shunted hepatectomized rats (n---4).It is therefore proposed that it is the amount of DP entering the liver which determines the production of CO2 and that a large part of DP is transformed extrahepatically into metabolites not subjected to further demethylation. This transformation takes place in the gut since the CO2 production was equivalent to that obtained after IV (11.5 5. 2.6%; n=6). In vitro microsomes of hepatic but not of intestinal origin generated demethylation to form a quantifiable amount of norpmpoxyphene. In conclusion, this study suggests that the prchepatic transformation of a drug may influence not only quantitatively but also qualitatively the production of its metabolites.
563
247A
564
G L U T A T H I O N E AS AN I M P O R T A N T ANTIOXIDANT AGAINST HYDROC, EN P E R O X I D E - M E D I A T E D DECRAESE IN MITOCHONDRIAL ENERGIZATION IN RAT HEPATOCYTES TREATED WITH ETHANOL. H Higuchi, I Kurose, N Watanabe, M Takaishi, S Kato, S Miura, and H lshii. Depl. qf Internal Med., Keio
lhJiv. Schmd ~!lMed. fbkyo 160. JAPAN. Therc has hccn much controvcrsy oil the clhanol induced impairment of oxygcn utili/ation. The present study was designed to evaluate the mechanism bx which cthanol modulates mitochondrial function in hepatocytes. Fo Ihat cfl'cct, hepatocytes were isolated from male Wistar rats, and cultured. After loading with either rhodamine 123 (Rh123), a mitochondrial membrane potential-sensitive fluorescence probe, or dichlorofluorescin 0)CF), a hydrogen peroxide-sensitive fluorescence probe, tat hepatocytcs were incubated with ethanol (50 raM). Alterations of Rhl23- and DCF-associated fluorescences in each hepatocyte were visualized under a laser scanning confocal microscope. The Rh123 fluorescence decreased to 40% and DCF fluorescence increased to 200% of the baseline valuc in each hepatocyte 30 minutes after incubation with ethanol. The decrease in Rh123 fluorescence and increase in DCF fluorescence observed after incubation with ethanol were significantly attenuated by addition of either N,N'-dimethylthiourea (10 raM), a cell membrane permeable hydrogen peroxide scavenger, or 4-methylpyrazole (500 l.tM). In contrast, 3-amino-l,2,4-triazole (1 mM), a catalase inhibitor, and maleic acid diethyl ester (MADE, 500 pM), a glutathione depictor, enhanced the ethanol-induced reduction of Rh123 fluorescence and elevation of I)CF fluorescence in hepatocytes. MADE was morepotent than ATZ to enhance these ethanol-induced fluorographic alterations. In some experiments, monochlorhimane (MCLB, 20 ~tM), a glutathionc-scnsitivc fluorescence probe; was added to the culture medium after each Rh123 study. This 2-color fluorescence study revealed that there is an inverse linear correlation between the glutathione content and the degree of ethanol-induced decrease in Rh123 fluorescence m an individual hepatocytes. Thus, the present study suggests that imracellular glutathione is an important cytoprotective factor against hydrogen peroxide-induced reduction of mitochondrial energization in ethanol-treated hepatocytes.