The glutathione S-transferases (GSTs) are a group of homo- and heterodimeric multifunctional proteins which catalyze the conjugation of reduced glutathione to electrophilic molecules generated during the metabolism of xenobiotics. The GSTs also detoxify lipid hydroperoxides, hydroperoxide-residues on DNA, participate in physiologic processes (prostaglandin isomerase) and may have a role in nuclear regulatory functions since one form has been found to be bound to DNA. Pyrrole, pyrrolidine, gamma- picoline and piperidine are used extensively in research laboratories, chemical and pharmaceutical industries and in the manufacture of textiles, rubber, dyes and explosives. Relatively little is known regarding their effects on cell function or metabolic processes despite the facts that man actually secretes between 3 and 20 mg of piperidine per day. Preliminary results in our laboratory reveal that pyrrole, pyrrolidine, gamma-picoline and piperidine induce GST activity to an extent greater than or equal to that produced by either phenobarbital or 3-methylcholantrene i.e. 1.7 to 2.5-fold). SDS-PAGE of liver cytosal of treated animals reveals the presence of protein bands of enhanced intensity migrating in the region of the three major subunits (Ya, Yb, Yc) present in rat cytosol; for treated rabbits SDS-PAGE shows the presence of new bands in the region of the GSTs. Thus, research is proposed to: 1) isolate and purify the individual GST isoenzymes from pyrrole, pyrroliding, gamma-picoline or piperidine induced rats and rabbits; 2) produce polyclonal antibodies to synthetic polypeptides of the subunits or monoclonal antibodies to the GSTs; 3) quantify GST isoenzyme hetero- and homodimer levels using an immunochemical """"""""sandwich"""""""" assay; 4) monitor gene activation (poly (A+) mRNA levels) by the nitrogen heterocycles; 5) examine transcriptional activation of the GST genes using the nuclear run on assay, gel and Northern blots; and 6) develop a """"""""matrigel"""""""" hepatocyte cell culture system for examination of GST induction by the aforementioned agents in vitro. Techniques such as FPLC, 1- and 2-D SDS-PAGE, isoelectric focusing, ELISA. Western and Northern blots. RNA polysome isolation and cell free translation will be used to characterize the induced GSTs and mechanism(s) of GST induction. This research will provide information on the GSTs induced by the nitrogen heterocycles and regulatory mechanism(s) which govern GST induction-an area of considerable importance given the multifunctional role of the GSTs.
