Histological Detection of Lipid Peroxidation Following Infusion of Tert-Butyl Hydroperoxide and ADP-Iron Complex in Perfused Rat Livers.

Abstract

ABSTRACT-Lipid peroxidation was assessed histologically and biochemically in hemoglobin-free perfused rat livers using two different types of stimulators. The Schiff reaction of fuchsin with cellular aldehydes was used as a histological index for lipid peroxidation. t-Butyl hydroperoxide (BHP, 0.8 mM) infusion caused a rapid and sus tained release of thiobarbituric acid reactive substances (TBARS) into the effluent perfusate for up to 60 min, which was accompanied by lactate dehydrogenase (LDH) leakage after 30 min. The Schiff positive foci were initially restricted to periportal zones and spread with time to whole areas, accompanied by periportal necrosis. Co infusion of diphenyl-p-phenylenediamine suppressed the TBARS release, with nega tive fuchsin staining, but the LDH leakage was unaffected. Under retrograde perfu sion, BHP produced pericentral staining and necrosis. With 2.5 mM ADP-100'U M Fe3+, little TBARS was released up to 60 min, even though the hepatic TBARS levels increased considerably by this time. By 90 min, marked TBARS release occurred, but LDH leakage remained low. Irrespective of the direction of perfusion, pericentral hepatocytes became Schiff positive after 30 min. The fuchsin staining method may be useful for detecting peroxidized zones of the liver lobules.

Histological Evidence for Dissociation of Lipid Peroxidation and Cell Necrosis in Bromotrichloromethane Hepatotoxicity in the Perfused Rat liver.

“…The method of surgery and the perfusion apparatus were described elsewhere (7,15). The perfusion was conducted in a non-recircu lating, constant flow rate (25 ml/min) system in either an antero or retrograde manner.…”

“…Small blocks of the pre-fixed liver were cut into approximately 50 u m-thick slices in cold 0.1 M phosphate buffer (pH 7.4) containing 20% glycerin, by using a tissue microslicer (Dosaka EM, Japan). Fuchsin staining was conducted essentially according to the previously de scribed method (7,15). Necrotic areas of the liver lobules were directly examined in 50,um thick slices of trypan blue-stained and pre-fix ed livers, with or without fuchsin co-staining.…”

“…The TBARS content and LDH activity in the effluent perfusate were measured as de scribed previously (7,15). …”

“…Since aldehydes are formed in the in vitro peroxidation system of microsomal lipids and also in vivo early after intoxication of rats with CC14 and CBrCl3 (11)(12)(13)(14), the Schiff-positive reaction may be considered as one piece of evidence for lipid peroxidation in halogeno methane hepatotoxicity. In the previous study (15), by using tert-butyl hydroperoxide and ADP-iron complex as lipid peroxidation stimu lators, we have shown that this staining method is useful for the identication of perox idized lobular zones in isolated perfused livers.…”

K+-Linked Release of Oxidized Glutathione Induced by tert-Butyl Hydroperoxide in Perfused Rat Liver Is Independent of Lipid Peroxidation and Cell Death.

“…This disturbance in the re dox balance of the cellular glutathione state and an efflux of GSSG is generally considered to be an index of oxi dative stress (10). On the other hand, BHP induces lipid peroxidation in animals (11), isolated livers (12,13) and cultured hepatocytes (14,15). Hydroperoxides also in duce microsomal lipid peroxidation in vitro (16,17).…”