Neil Kaplowitz

Role of TNF‐α in ethanol‐induced hyperhomocysteinemia and murine alcoholic liver injury

We previously reported a link between ethanol‐induced elevation of homocysteine, endoplasmic reticulum (ER) stress, and alcoholic liver injury in the murine model of intragastric ethanol feeding. We studied the role of TNFα in this setting by using TNFR1 knockout mice (C57 BL/6). There was a 7.4‐fold increase of homocysteine in wild‐type and a 6‐fold increase in TNFR1 knockout mice with intragastric alcohol exposure for 4 weeks. Plasma TNFα increased in the wild‐type (18.4 ± 3.3 pg/mL vs. 8.4 ± 1.3 pg/mL (control)) and in the knockouts (12.9 ± 1.4 pg/mL vs. 7.2 ± 1.6 pg/mL (control)). Similar extent of fatty liver was observed in both types. Increased ALT was observed in both groups. Necroinflammatory foci were increased significantly in ethanol‐fed knockouts but not to the same extent as in the ethanol‐fed wild type. Increase of hepatic apoptosis and reduction of S‐adenosyl‐L‐methionine was detected in both types of animals fed ethanol. ER stress demonstrated by RT‐PCR of mRNA of selective ER stress markers GRP78, CHOP, and SREBP1 was increased equivalently in both types of mice. Betaine administration decreased ER stress in conjunction with attenuation of the elevated plasma homocysteine in both types of animals. Betaine increased hepatic S‐adenosyl‐L‐methionine by 28 fold in the knockouts and by 24‐fold in wild type. In conclusion, TNFα makes a moderate contribution to the ALT elevation, necroinflammation, apoptosis, a small contribution to the fatty liver and no contribution to hyperhomocysteinemia and ER stress in intragastric alcohol fed mice. (HEPATOLOGY 2004;40:442–451.)

Binding of bile acids, oleic acid, and organic anions by rat and human hepatic Z protein

Binding affinities of purified Z proteins from rat and human liver for bile acids, oleic acid, and organic anions were studied. Purification of Z protein from both rat and human hepatic cytosol was performed by gel filtration, chromatofocusing, and hydroxyapatite chromatography. Both purified proteins showed the same molecular weight (Mr = 14,000) and isoelectric points were 6.9 and 6.5 for rat and human proteins, respectively. Binding studies were performed by the competitive displacement of 1-anilino-8-naphthalene sulfonate. Rat and human Z proteins exhibited similar binding affinities for bile acids, oleic acid, and organic anions. Among various bile acids, both proteins bound monohydroxy bile acids with high affinity and trihydroxy bile acids with low affinity; sulfates were bound with higher and glucuronides with lower affinity than their parent bile acids. In comparison with GSH S-transferases, rat Z protein had lower affinity for bile acids than rat GSH S-transferase B and human Z protein had higher affinity for bile acids than human cationic GSH S-transferase. The role for Z protein in the intracellular binding of bile acids may be particularly important in human liver.

Binding of ethacrynic acid to hepatic glutathione S-transferases in vivo in the rat

Abstract Ethacrynic acid is a substrate for rat glutathione S -transferases in vitro (1,2). In their study defining the importance of the glutathione S -transferases in the hepatic metabolism and biliary excretion of ethacrynic acid, Wallin et al . (3) suggested that a small but consistent portion of the administered drug was bound selectively and covalently to the transferases. Such an association would represent a unique example of covalent binding of a drug to the metabolizing enzyme without prior microsomal enzyme activation. We have undertaken these studies, therefore, to characterize the binding of ethacrynic acid to the glutathione S -transferases and to delineate the selectivity of this binding.

Identification and purification of a 36 kDa bile acid binder in human hepatic cytosol

We recently purified two closely related 33 kDa proteins from rat hepatic cytosol, designated bile acid binder I and II, which selectively bind bile acids with comparable affinity as glutathione S‐transferase B. This work has now been extended to human liver in which we have identified a similar cytosolic binding activity in the 30–40 kDa fraction from gel filtration. Subsequent chromatofocusing and hydroxyapatite chromatography resulted in the isolation of a homogenous monomeric protein of 36 kDa. The binding affinity of this protein for lithocholate using the displacement of 1‐anilino‐8‐naphthalenesulfonate (ANS) was 0.1 μM, whereas human hepatic glutathione S‐transferases purified from glutathione affinity chromatography demonstrated no competitive displacement of ANS.

Newly identified organic anion-binding proteins in rat liver cytosol

Previously, all organic anion binding activity in the Y protein (ligandin-containing) fraction of rat liver cytosol has been attributed to the glutathione S-transferases. Gel filtration on Sephadex G-75 superfine has resolved the Y protein fraction into organic anion-binding fractions of two distinct molecular weights: (a) Mr 45 000 containing the glutathione S-transferases, and (b) Mr 35 000 referred to as Y. Two proteins (Dv and D1) were purified from the Y fraction. Dv is a basic protein consisting of four subunits of Mr 8000-9000 and selectively binds 1-anilino-8-naphthalene sulfonate and sulfobromophthalein. D1 is a monomer (Mr 40 000) which exhibits high affinity binding for Rose Bengal. Dv protein bound a broad spectrum of organic anions. Antibody raised in rabbits to rat Dv showed no cross-reactivity with purified glutathione S-transferases A, B, or C. Thus, we have identified organic anion-binding proteins which are unrelated to the glutathione S-transferases, but which were previously associated with the crude Y-fraction. The relative abundance of these proteins and their binding characteristics suggest their role in hepatic organic anion transport.

Oxidation and reduction of bile acid precursors by rat hepatic 3α-hydroxysteroid dehydrogenase and inhibition by bile acids and indomethacin

Enzyme kinetics of purified rat hepatic 3 alpha-hydroxysteroid dehydrogenase for bile acid precursors and effects of bile acids and indomethacin on those activities were studied. This enzyme catalyzed the oxidoreduction of the C3 position of bile acid precursors. Km for 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one (1.6 microM) was markedly lower than Km for 7 alpha-hydroxy-5 beta-cholestan-3-one (28 microM) but Vmax was similar. Km for 3 alpha, 7 alpha-dihydroxy-5 beta-cholestane (12 microM) was lower than Km for 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestane (150 microM) although Vmax/Km values were similar for both compounds. Bile acids and indomethacin inhibited the reduction of 3-oxo bile acid precursors. NADPH inhibited the binding of lithocholic acid (3 alpha-hydroxy-5 beta-cholanic acid) by 3 alpha-hydroxysteroid dehydrogenase. These data suggest that intrahepatic bile acid concentrations may affect the reduction of 3-oxo-bile acid precursors and intrahepatic redox conditions may affect intracellular bile acid transfer.

Tocopherol-Binding Proteins of Hepatic Cytosol

Tocopherol is a highly lipophilic substance. It is believed to travel in the circulation nonspecifically bound to lipoproteins. The liver is believed to play an important role in the processing of dietary aand y-tocopherol. The transfer of tocopherol between intracellular compartments in the hepatocyte is believed to involve cytosolic proteins that bind tocopherol. Catignani in 1975 first described a molecular weight 31,000 fraction in gel filtration of rat liver cytosol that bound tocopherol. A void volume fraction was also noted to bind labeled a-tocopherol. Excess a-tocopherol, however, displaced the labeled form only in the 31,000 molecular weight fraction. Subsequently it was shown by Catignani and Bieri that 400-fold excess a-tocopherol displaced 98% of the label, but y-tocopherol displaced 60% of the label.* The binder was identified only in the liver and was found in mouse, guinea pig, rabbit, hamster, and chicken in addition to rat liver. Studies of specificity of binding revealed the need for free chroman hydroxyl group, intact chromanol ring, and side chain? Three groups subsequently demonstrated the capacity of the crude cytosol fraction to transfer tocopherol. Murphy and Mavis demonstrated the activity of a 34,000 molecular weight gel filtration fraction from rat liver to transfer tocopherol from egg phosphatidylcholine liposomes to liver microsome^.^ The activity was present only in liver and was absent in lung, heart, and brain of rats. Mowri et al. demonstrated the ability of this rat liver fraction to transfer a tocopherol from liposomes to microsomes? They showed that excess a-tocopherol could displace labeled a-tocopherol; y-tocopherol also exhibited some inhibitory effect. They claimed to observe this activity in liver, spleen, heart, and lungs but not in kidney or brain cytosol of the rat. They also claimed that human liver possessed this activity but showed no data. Behrens and Mad&e showed that labeled tocopherol was associated with a high molecular weight peak and a 32,000 molecular weight fraction in gel filtration of hepatic cytosol after oral dosing in vivo with labeled a-tocopherol.5 Subsequently they partially purified the fraction from rat liver, using ammonium sulfate fractionation, gel filtration, and ion exchange chromatography and showed transfer of labeled tocopherol from the binding protein to microsomes.6 Their studies suggested that the molecular form of endogenous tocopherol associated with the void volume was predominantly y-tocopherol, whereas the molecular form associated with the

Role of Glutathione Status in Protection against Ethanol-Induced Gastric Lesions

The role of glutathione status in gastric mucosal cytoprotection has been a subject of controversy. Cysteamine, an exogenous sulfhydryl agent and diethyl maleate (DEM), an endogenous glutathione (GSH) depletor both appear to protect rats from ethanol-induced gastric lesions. In this study, we used various agents to alter gastric mucosal GSH levels and assessed the effects on susceptibility to ethanol injury. We found that DEM and buthionine sulfoximine both depleted gastric GSH but only DEM protected against ethanol-induced gastric lesions. L-Oxothiazolidine-4-carboxylate (OXT) and N-acetyl-L-cysteine (NAC) both potentiated ethanol-induced gastric lesions even though only NAC significantly raised the GSH level. The depletion of GSH by DEM was reversed by supplying cysteine in the form of OXT or NAC so that the net result was a GSH level close to normal control. The potentiation of ethanol injury by NAC and OXT was still apparent. These experiments show no relation between gastric GSH levels and susceptibility to ethanol injury.

Trans-stimulation and driving forces for GSH transport in sinusoidal membrane vesicles from rat liver

Sinusoidal membrane vesicles from rat liver were employed to study the characteristics of GSH transport. Saturable concentration dependent uptake was best described by the sum of a high and low Km transport. Preloading with GSH markedly stimulated the initial uptake of GSH. GSH transport was electrogenic; uptake was enhanced by an inwardly directed K+ gradient which could be blocked by the K+-channel blocker, Ba2+. The other cations such as Na+, Li+ were poor substitutes for K+. These results therefore show that net GSH transport involves movement of K+.

Effect of Phorone and Allopurinol on Ischemia-Reperfusion Injury in Gastrointestinal Mucosa of the Rat

We studied the effect of inhibition of oxyradical formation and of endogenous glutathione (GSH) depletion on lesion formation in the gastrointestinal tract in a modified rat hemorrhagic shock model (1 h hypotension and 1 h reperfusion). Allopurinol, an inhibitor of xanthine oxidase, did not protect against lesion formation. This suggests that oxygen radicals generated from xanthine oxidase may not be the major cause of injury under these conditions of prolonged ischemia-reperfusion. Phorone (diisopropylideneacetone), a GSH depletor, decreased mucosal GSH levels in the corpus, duodenum and small intestine, and also significantly reduced lesion formation histologically in the corpus, antrum, duodenum and small intestine. However, there was no significant differences in mucosal blood flow (as estimated by changes in mucosal hemoglobin concentrations and oxygen saturation of mucosal hemoglobin) in the corpus, antrum, duodenum and small intestine between phorone-pretreated and control rats. We conclude that phorone decreased mucosal GSH concentrations and exerted a protective effect against hemorrhagic shock-induced gastrointestinal mucosal lesions. The protective effect appears to be independent of mucosal blood flow.