Cultures of rat hepatic cells will be employed to investigate the role of two vitamins in liver function. These are vitamin E and folic acid. Vitamin E has been proposed to function by quenching free radicals which are generated primarily as a result of oxygen metabolism. The presence of excessive amounts of free radicals in tissues is believed to result in physiological deterioration which is manifested by aging, coronary disease, connective tissue disorders and tumorogenesis. We will examine the rapid vitamin E, glucocorticoid-reversible membrane dysfunction occurring when hepatocytes are placed in culture in order to further understand the role of vitamin E in these processes. The hepatocyte membrane instability will be evaluated by the loss of two liver specific transport systems: one which transports asialoglycoproteins and one which is an anion transport system which is involved in cellular uptake of bromosulfophthalein, cholic acid, and methotrexate. The investigations are designed to determine the reasons for the loss of these transport systems and way in which vitamin E stabilizes them. The liver functions as a storage site for folates. In order to be retained by tissues folates must be converted to Gamma-glutamyl derivatives. Hepatocyte culture systems will be used to investigate the synthesis and cleavage of these derivatives and the factors which regulate these processes. Permeabilized hepatocytes and hepatoma cells have been developed to study the Gamma-glutamylation reaction in situ. Cells which have been depleted of the folylpolyglutamate coenzymes will be employed to study the Gamma-glutamylation of folates and antifolates and its regulation. These studies will be designed to understand what factors and conditions control the storage and release of folate coenzymes in liver and what effects the antifolates utilized in cancer chemotherapy may have on this process. Emphasis will be placed upon alterations in glutamylation caused by insulin, glucocorticoids, and glucagon and also the growth state and folate content of the cells.
