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Australia antigen was found in clams contaminated by drainage of untreated sewage from a coastal hospital. In closed-system aquariums, the antigen was ingested by clams and transmitted to previously uninfected clams. In opensystem aquariums, the titer of Australia antigen decreased with time, suggesting viral concentration rather than replication.

Evidence is presented that ligandin, an intracellular protein involved in the binding of such anions as bilirubin, indocyanine green, and penicillin, is identical to glutathione S-transferase B (EC 2.5.1.18), an enzyme catalyzing the conjugation of glutathione with such electrophiles as 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, iodomethane, ethacrynic acid, and bromosulfophthalein. The proteins, isolated by distinct methods, have the same specificity for substrates and for ligands, react in identical fashion to antibody produced against ligandin, bear entirely similar physical characteristics and amino acid composition, and are both induced in response to phenobarbital. Indocyanine green, one of the ligands that is not effective as a substrate, was shown to competitively inhibit the conjugation reaction. It is suggested that specificity is directed toward compounds with electrophilic sites.

ALTHOUGH THE LIVERS OF RATS TREATED WITH AGENTS KNOWN TO SUPPRESS BILIRUBIN TRANSPORT APPEARED RELATIVELY NORMAL ON ROUTINE HISTOLOGICAL SECTIONS, CYTOCHEMICAL AND ELECTRON MICROSCOPIC PREPARATIONS REVEALED MARKED CHANGES IN: (a) levels of alkaline phosphatase and apparent ATPase activities of the cell membrane at the sinusoids, bile canaliculi, and between adjacent cells; (b) morphology of the bile canaliculi such as dilatation, fragmentation, vesiculation and reduced numbers of microvilli; (c) the number of lysosomes and their distribution within the cell. Similar changes in the cytochemistry of the liver cell were induced by extrahepatic obstruction.

An antibody produced against rat Y protein, the major cytoplasmic organic anion-binding protein in liver, was characterized. The antibody precipitated Y protein from liver supernatant fractions and specifically removed the organic anion-binding capacity from this fraction.Y protein was detected by immunodiffusion with this antibody in the supernates of rat liver, kidney, and small intestinal mucosa and was not detected in supernates of 16 other tissues including bile and serum. Precipitation with anti-Y was not detected with supernates of liver from 20 other species, including man. Quantitative radial immunodiffusion revealed Y protein to constitute 4.5% of supernatant protein in rat liver and approximately 2% of supernatant protein in rat kidney and small intestinal mucosa. Phenobarbital administration increased the concentration of Y protein in rat liver by 280%, but not in kidney or small intestinal mucosa, and was associated with increased plasma disappearance of sulfobromphthalein sodium, indocyanine green, and bilirubin, and increased hepatic, but not kidney or small intestinal mucosal, content of these organic anions. These observations provide further evidence indicating that the concentration of Y protein is a major determinant of organic anion flux across the plasma membrane of the liver cell.Immunodiffusion and immunoelectrophoresis revealed serological identity between Y protein, cortisol metabolite-binding protein I. and the major azocarcinogen-binding protein.

The process by which various anions, including bilirubin and several dyes, drugs, hormones and their metabolites, are transferred from plasma into the liver cell is poorly understood. Two hepatic cytoplasmic proteins, Y and Z, that bind various organic anions in vivo and in vitro have been postulated to be involved in this process. The concentration of Y, the major organic anion-binding protein, increases in rat liver after administration of phenobarbital in association with enhanced organic anion transfer from plasma into liver as determined by initial plasma disappearance rate (K(1)) and hepatic dye content for sulfobromophthalein (BSP) and indocyanine green (ICG), as well as increased relative hepatic storage of BSP. Acute bile duct ligation failed to alter plasma disappearance or hepatic content of BSP in normal or phenobarbital-treated rats. Other drugs and chemicals which cause proliferation of hepatic smooth endoplasmic reticulum and enhancement of drug metabolism, such as allylisopropylacetamide, dieldrin, DDT, 3-methylcholanthrene, and benzpyrene increased Y and BSP K(1) and, where studied, hepatic BSP content. Alcohol feeding had no effect on Y, Z, or K(1) for BSP. Hypophysectomy and thyroidectomy increased Y but decreased K(1) and, where studied, hepatic content of BSP. Of several hormones studied, only thyroxine restored Y and K(1) to normal in hypophysectomized or thyroidectomized rats. Mice with congenital pituitary insufficiency also manifested increased Y which returned to normal after thyroxine administration. In hormone-deficient rats and mice, phenobarbital administration produced a further increase in Y suggesting that different mechanisms may be responsible for the change in Y resulting from drug administration and hormonal deprivation. Thyroxine, testosterone, or hydrocortisone did not alter BSP K(1) or Y in normal rats.Cholestasis produced by ethinyl estradiol administration or biliary obstruction reduced Y, Z, BSP K(1) and hepatic BSP content. These results support the hypothesis that Y and Z are involved in the transfer of BSP, ICG, and possibly other organic anions from plasma into the liver. The concentration of Y increased after administration of various drugs and chemicals as well as in thyroid deficiency. Thyroid hormone appears to be important in regulation of the intracellular concentration of Y. Because thyroid deficiency increased Y but decreased BSP K(1) and hepatic BSP content, other factors beside Y and Z influence hepatic organic anion uptake.

Previous studies based upon competition between different organic anions for biliary excretion in vivo have suggested that all organic anions share a common hepatic secretory mechanism. Corriedale sheep with an inherited defect in organic anion excretion by the liver were used to study this problem directly without the need for competition studies, the results of which are difficult to analyze. Maximal biliary excretion of sulfobromphthalein (BSP) in mutant Corriedale sheep was less than 7% of that observed in normal sheep whereas maximal biliary excretion of taurocholate, the major organic anion in sheep bile, was not different in mutant and normal sheep. Taurocholate infusion enhanced maximal hepatic excretion of BSP in normal but not in mutant sheep. These studies of an inheritable disorder which appears to be identical to the Dubin-Johnson syndrome in man, demonstrate that taurocholate excretion requires at least one step in biliary excretion which is not required by other organic anions such as bile pigment, porphyrins, drugs, and dyes.

The administration of phenobarbital to rats enhanced hepatic uptake of an organic anion, bromsulphalein, in vivo and simultaneously increased the amount of Y, a hepatic cytoplasmic organic anion-binding protein. This study supports the postulate that Y is a major determinant in the selective hepatic uptake of certain organic anions from plasma. Induction of Y may contribute to the enhanced hepatic uptake and metabolism of various organic anions (drugs, hormones, etc.) produced by phenobarbital and other agents.