Our objectives are to elucidate the cause and effect chain of molecular events elicited by the model acute hepatotoxin CCl4. We propose a series of multifaceted but highly integrated investigations in fasted, phenobarbital pretreated rats. Primary events, i.e., the volatile, soluble and covalently bound metabolites of CCl4, will be defined using chromatographic analysis (including GC-MS) plus radiolabeled (14C and 36Cl) compound and histoautoradiography. Secondary events, i.e., the molecular aberrations of composition and structure (including products of lipid oxidation, levels of nucleotides and antioxidants, and the contents and distribution of metals), will be defined using GC, TLC, HPLC, atomic absorption and electron microprobe techniques. Tertiary events, i.e., the changes in cell function and structure will be defined using histochemistry, isolation of selectively affected enzyme components (including G-6-Pase, cytochrome P-450 and ribosomes), electron microscopy (including scanning and freeze fracture), and immunohistochemistry. We propose to evaluate the relevance of these molecular events to the injurious process elicited by CCl4: first, by time-course and dose-response studies; second, by complementary studies of prevention of biochemical events through intervention with the protective agent cystamine; and fourth, study of molecular events associated with CCl4 injury in the more manipulatable and accessible isolated hepatocytes in cultures.
