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Effect of Norepinephrine (NE) on GSH export by hepatic and extra-hepatic tissues into plasma and bile in vivo
I COMPARATIVE STUDY ON ANTIOXIDANT ACTIVITY OF ALPHA- AND GAMMA-TOCOPHEROLS IN MOUSE BRAIN HOMOGENATES H Noriko Noguchi and Etsuo Niki, RCAST, Llniverszty of Tokyo, &$%%I4
Effect of Norepinephrioe (NE) on GSH Export by Hepatic and Extra-hepatic Tissues into Plasma and Bile in viva. Z. Son% K. Beers, and W. Bottje. Dept. of Poultry Science, University of Arkansas, Fayetteville, AR 72701 Few in viva investigations of hepatic export of GSH have been
/APAN.
Membrane antioxidant vitamin E consists of two major forms, alpha-tocopherol (A-TOH) and gamma-tocopherol (G-TOH). A-TOH has been well known as the most potent lipophilic antioxidant in viva. There is increasing interest that G-TOH may act efficiently in scavenging peroxynitrite, a powerful mutagenic oxidant and nitrating species. We have studied the antioxidant effect of A-TOH and G-TOH against peroxyl radical and peroxynitrite in homogenates of mouse brain without A-TOH, which enables us to incorporate A- or G-TOH to the homogenates by the same way. A-TOH was found to be more effective than G-TOH in inhibiting lipid peroxidation induced by peroxyl radical generated from 2,2’-azobis (2-amidinopropane) dihydrochloride. However, G-TOH was more powerful in decreasing lipid peroxidation induced by peroxynitrite generated from 3-morpholino-sydnonimine. A-TOH was depleted more rapidly than G-TOH in the brain homogenates induced by peroxynitrite whether they were added separately or together to the homogenates. Interestingly, most of the oxidation product of A-TOH by peroxynitrite was alpha-tocopheryl quinone, while over 50 percent of G-TOH oxidized by peroxynitrite was transformed to nitro-gamma-tocopherol (NGTH). In addition, it is known that A-TOH and G-TOH react with a hydrogen-abstracting oxidant, galvinoxyl, stoichiometrically. However it was found that NGTH was very stable and did not react with galvinoxyl although it possesses the same hydroxyl group as G-TOH.
reported in mammals. In this study, an in viva model in birds (which lack a diaphragm) was used to determine the effect of NE on GSH levels in the hepatic vein, hepatic portal vein, and bile duct in male single comb white leghorn chickens. In an initial experiment, basal biliary reduced (GSH) and oxidized (GSSG) glutathione secretion rates were 4.6 f 2.1 and 5.9 + 0.5 nmol/min/kg BW, respectively, and increased by -100% following inhibition of y-glutamyl transpeptidase activity by retrograde infusion of AT-125. In Exp. 2, cannulae were placed in the carotid artery, hepatic vein, hepatic portal vein and bile duct. NE (2 and 10 pg/min/kg BW) was infused into the hepatic portal vein for 20 min. Blood and bile samples were collected before, during and after each NE infusion. The 2 pg NE/min infusion had no affect on blood pressure, but elevated hepatic venous GSH. The 10 Kg NE/min infusion raised hepatic and portal venous GSH, and systemic and hepatic pressures. NE had no effect on bile flow, or biliary GSH, GSSG and total GSH, however. Phentolamine (an a-receptor antagonist) blocked hepatic export of GSH and NE-stimulated increases of plasma GSH in the hepatic and hepatic portal veins. The results demonstrate that NE stimulated GSH export by both hepatic and extra-hepatic tissues via an areceptor mediated mechanism in viva, but did not affect biliary GSH secretion. The avian model used in this study may be useful in
understanding other aspects of hepatic regulation of GSH and other compounds in viva.
Ill1
1110I
I
LOCALIZATION OF IMhJUNOHISTOCHEMICAL METALLOTHIONEIN IN RAT LIVER, KIDNEY AND AFTER DIETARY ZINC lNTESTINF! SMALL Carla Tavlor & Elzbieta Szczurek. Foods MANIPULATIONS. & Nutrition Dept., Univ. of Manitoba, Winnipeg, MB, R3T 2N2. Metallothionein (MT) may protect against cellular damage caused by chemical stress by donating hydrogen ion and/or releasing zinc (Zn) for cellular repair. To tirther understand the interaction between Zn status and MT, we studied the effect of dietary Zn deficiency and repletion on MT localization in rat liver, kidney and small intestine using immunohistochemical staining. Weanling Sprague Dawley rats were fed Zn deficient (ZD), pair-fed (PF) and control (C) diet for 3 wks. Half of ZD and PF animals were repleted with the control diet (ZR and PFR groups) for additional 24 hours. In small intestine, strong staining of Paneth cells was observed for all groups with the exeption of ZD. Surface epithelial cells stained in the intestine of ZR, PF & C rats. In liver, strong staining of hepatocytes was demonstrated for PF & PFR animals. The staining was week in C and absent in ZD and ZR. In kidney of AL, PF and PFR, epithelial cells of proximal and distal tubules showed strong staining, as opposed to moderate and weak staining in ZR and ZD, respectively. The epithelia of large collecting ducts stained intensely in kidney of all animals. The observed changes in MT localization in response to dietary Zn may explain site-specific differences in tissue and cellular damage due to chemical and oxidative stress.
s44
109
OXYGEN
I
I
EFFECT OF DIETARY ZINC ON METALLOTHIONEIN(MT) EXPRESSION AND TUMORIGENESIS w Kfll nnn Ghoshal, Samson T Jacob and Tammy M. Bray, The Ohio State University.
P olumbus
OH
MT, a low molecular weight metal binding protein consists of 20 cycteines and usually binds 7 zinc atoms. MT expression has been linked with carcinogenesis, resistance to cancer therapy and tumor progression. MT responds to acute oxidative stress and is proposed to be an antioxidant. Zn has a high affinity to bind MT-I and MT-II and have the ability to regulate MT synthesis at the transcriptional level. In this study, we investigated the role of dietary Zn in MT expression related to tumor growth by implanting hepatorna cells in the rear limbs of rats. Immediately following implants, rats (n=12) were fed a Zn deficient (0 ppm, Zn-), Zn normal (100 pprn, Zn+) or Zn excess (3500 ppm, Zn++) diet for 27 days. The results showed that fhe tumor size was significantly higher in Zn++ group when compared to that of Zn+ or Zn-. Rats fed a Zn- diet have the typical lower concentration of serum Zn (0.2 ug/ml) compared to Zn+ (1.5 ug/ml) and Zn++ (1.9 @ml) groups. Zn level in hepatoma in the Zn++ group (30 ug/g) was significantly higher than that of Zn- (17 ug/g) or Zn+ (17 ug/g). However, Zn level in the limb muscle surrounding the hepatoma was significantly lower than that in hepatoma of all groups. Regardless of the high level of Zn, MT transcription was not detected in the hepatoma of all rats. In addition, there is no difference in DNA binding activity of MT transcriptional factor-l (MTFl) in hepatoma among all
groups. Interestingly, there was no detectable MT (total RNA) in the liver of rats fed the Zn- diet while the liver concentration of Zn was similar to that of rats fed Zn+. Feeding Zn++ diet increased (5 folds) Zn level in the liver and induced MT transcription and MTFl binding activity. The results suggest that tumor growth may have a high requirement for Zn.
’ 9
9
Effect of Norepinephrioe (NE) on GSH Export by Hepatic and Extra-hepatic Tissues into Plasma and Bile in viva. Z. Son% K. Beers, and W. Bottje. Dept. of Poultry Science, University of Arkansas, Fayetteville, AR 72701 Few in viva investigations of hepatic export of GSH have been
/APAN.
Membrane antioxidant vitamin E consists of two major forms, alpha-tocopherol (A-TOH) and gamma-tocopherol (G-TOH). A-TOH has been well known as the most potent lipophilic antioxidant in viva. There is increasing interest that G-TOH may act efficiently in scavenging peroxynitrite, a powerful mutagenic oxidant and nitrating species. We have studied the antioxidant effect of A-TOH and G-TOH against peroxyl radical and peroxynitrite in homogenates of mouse brain without A-TOH, which enables us to incorporate A- or G-TOH to the homogenates by the same way. A-TOH was found to be more effective than G-TOH in inhibiting lipid peroxidation induced by peroxyl radical generated from 2,2’-azobis (2-amidinopropane) dihydrochloride. However, G-TOH was more powerful in decreasing lipid peroxidation induced by peroxynitrite generated from 3-morpholino-sydnonimine. A-TOH was depleted more rapidly than G-TOH in the brain homogenates induced by peroxynitrite whether they were added separately or together to the homogenates. Interestingly, most of the oxidation product of A-TOH by peroxynitrite was alpha-tocopheryl quinone, while over 50 percent of G-TOH oxidized by peroxynitrite was transformed to nitro-gamma-tocopherol (NGTH). In addition, it is known that A-TOH and G-TOH react with a hydrogen-abstracting oxidant, galvinoxyl, stoichiometrically. However it was found that NGTH was very stable and did not react with galvinoxyl although it possesses the same hydroxyl group as G-TOH.
reported in mammals. In this study, an in viva model in birds (which lack a diaphragm) was used to determine the effect of NE on GSH levels in the hepatic vein, hepatic portal vein, and bile duct in male single comb white leghorn chickens. In an initial experiment, basal biliary reduced (GSH) and oxidized (GSSG) glutathione secretion rates were 4.6 f 2.1 and 5.9 + 0.5 nmol/min/kg BW, respectively, and increased by -100% following inhibition of y-glutamyl transpeptidase activity by retrograde infusion of AT-125. In Exp. 2, cannulae were placed in the carotid artery, hepatic vein, hepatic portal vein and bile duct. NE (2 and 10 pg/min/kg BW) was infused into the hepatic portal vein for 20 min. Blood and bile samples were collected before, during and after each NE infusion. The 2 pg NE/min infusion had no affect on blood pressure, but elevated hepatic venous GSH. The 10 Kg NE/min infusion raised hepatic and portal venous GSH, and systemic and hepatic pressures. NE had no effect on bile flow, or biliary GSH, GSSG and total GSH, however. Phentolamine (an a-receptor antagonist) blocked hepatic export of GSH and NE-stimulated increases of plasma GSH in the hepatic and hepatic portal veins. The results demonstrate that NE stimulated GSH export by both hepatic and extra-hepatic tissues via an areceptor mediated mechanism in viva, but did not affect biliary GSH secretion. The avian model used in this study may be useful in
understanding other aspects of hepatic regulation of GSH and other compounds in viva.
Ill1
1110I
I
LOCALIZATION OF IMhJUNOHISTOCHEMICAL METALLOTHIONEIN IN RAT LIVER, KIDNEY AND AFTER DIETARY ZINC lNTESTINF! SMALL Carla Tavlor & Elzbieta Szczurek. Foods MANIPULATIONS. & Nutrition Dept., Univ. of Manitoba, Winnipeg, MB, R3T 2N2. Metallothionein (MT) may protect against cellular damage caused by chemical stress by donating hydrogen ion and/or releasing zinc (Zn) for cellular repair. To tirther understand the interaction between Zn status and MT, we studied the effect of dietary Zn deficiency and repletion on MT localization in rat liver, kidney and small intestine using immunohistochemical staining. Weanling Sprague Dawley rats were fed Zn deficient (ZD), pair-fed (PF) and control (C) diet for 3 wks. Half of ZD and PF animals were repleted with the control diet (ZR and PFR groups) for additional 24 hours. In small intestine, strong staining of Paneth cells was observed for all groups with the exeption of ZD. Surface epithelial cells stained in the intestine of ZR, PF & C rats. In liver, strong staining of hepatocytes was demonstrated for PF & PFR animals. The staining was week in C and absent in ZD and ZR. In kidney of AL, PF and PFR, epithelial cells of proximal and distal tubules showed strong staining, as opposed to moderate and weak staining in ZR and ZD, respectively. The epithelia of large collecting ducts stained intensely in kidney of all animals. The observed changes in MT localization in response to dietary Zn may explain site-specific differences in tissue and cellular damage due to chemical and oxidative stress.
s44
109
OXYGEN
I
I
EFFECT OF DIETARY ZINC ON METALLOTHIONEIN(MT) EXPRESSION AND TUMORIGENESIS w Kfll nnn Ghoshal, Samson T Jacob and Tammy M. Bray, The Ohio State University.
P olumbus
OH
MT, a low molecular weight metal binding protein consists of 20 cycteines and usually binds 7 zinc atoms. MT expression has been linked with carcinogenesis, resistance to cancer therapy and tumor progression. MT responds to acute oxidative stress and is proposed to be an antioxidant. Zn has a high affinity to bind MT-I and MT-II and have the ability to regulate MT synthesis at the transcriptional level. In this study, we investigated the role of dietary Zn in MT expression related to tumor growth by implanting hepatorna cells in the rear limbs of rats. Immediately following implants, rats (n=12) were fed a Zn deficient (0 ppm, Zn-), Zn normal (100 pprn, Zn+) or Zn excess (3500 ppm, Zn++) diet for 27 days. The results showed that fhe tumor size was significantly higher in Zn++ group when compared to that of Zn+ or Zn-. Rats fed a Zn- diet have the typical lower concentration of serum Zn (0.2 ug/ml) compared to Zn+ (1.5 ug/ml) and Zn++ (1.9 @ml) groups. Zn level in hepatoma in the Zn++ group (30 ug/g) was significantly higher than that of Zn- (17 ug/g) or Zn+ (17 ug/g). However, Zn level in the limb muscle surrounding the hepatoma was significantly lower than that in hepatoma of all groups. Regardless of the high level of Zn, MT transcription was not detected in the hepatoma of all rats. In addition, there is no difference in DNA binding activity of MT transcriptional factor-l (MTFl) in hepatoma among all
groups. Interestingly, there was no detectable MT (total RNA) in the liver of rats fed the Zn- diet while the liver concentration of Zn was similar to that of rats fed Zn+. Feeding Zn++ diet increased (5 folds) Zn level in the liver and induced MT transcription and MTFl binding activity. The results suggest that tumor growth may have a high requirement for Zn.
’ 9
9