TRYPAN BLUE UPTAKE AS A NEW METHOD TO INVESTIGATE HEPATOTOXICITY IN PERIPORTAL AND PERICENTRAL REGIONS OF THE LIVER LOBULE - STUDIES WITH ALLYL ALCOHOL IN THE PERFUSED LIVER
A new method based on trypan blud uptake was developed to study zonal hepatotoxicity in specific regions of the liver lobule in perfused livers. Allyl alcohol (350 .mu.M), a hepatotoxin that causes necrosis of periportal regions, was infused into livers from phenobarbital-treated rats for 60 min. The subsequent infusion of trypan blue (0.2 mM) stained nuclei in periportal but not midzonal or pericentral regions of the liver lobule. With this new method of dye uptake, comparison of the temporal relationship between metabolic changes and the onset of cellular damage was possible. During the 1st 10 min of allyl alcohol infusion, allyl alcohol was taken up at rates of 64 .mu.mol/g per h concomitant with an increase in O2 uptake of 18 .mu.mol/g per h. Allyl alcohol infusion also increased bile flow from 69 to 167 .mu.l/min and released oxidized glutathione from 127 to 217 nmol/g per h, respectively. However, during the next 20-min phase of exposure to allyl alcohol, O2 uptake was inhibited by 60% and allyl alcohol uptake declined by 80%. Bile flow and release of oxidized glutathione also declined continously between 10 and 30 min and by 30 min stopped completely. Concomitant with the inhibition of O2 uptake, rates of glycogenolysis and glycolysis increased by 130 and 85%, respectively. Within minutes after maximal inhibition of O2 uptake, between 30 and 40 min after initiation of allyl alcohol infusion, trypan blue was taken up by hepatocytes in periportal zones of the liver lobule. Subsequently, lactate dehydrogenase began to appear in the effluent perfusate. Trypan blue staining of periportal zones of the liver lobule did not occur until significant alterations in metabolism were observed. Trypan blue uptake provides a new, rapid, convenient and inexpensive approach to define the time course of hepatotoxicity in distinct regions of the liver lobule.