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Structually diverse peroxisome-proliferators (PPs) were investigated regarding their effects on NAD+ level and two key enzyme activities in the tryptophan (Trp)-NAD+ pathway in the liver of rats (Sprague-Dawley male) fed PP-containing diets freely for 2 weeks. All PPs, except for thyroxine, significantly increased hepatic NAD+ level in concert with hepatic hypertrophy. Activity of quinolinate phosphoribosyltransferase (QAPRTase), one of the key enzymes in the Trp-NAD+ pathway, was increased by the PPs which caused significant increase in the hepatic NAD+. On the other hand, alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSDase), another key enzyme in the Trp-NAD+ pathway, was drastically inhibited by all PPs except for linolenic acid, which was only slightly inhibitory. Most PPs investigated activated peroxisomal marker enzymes such as palmitoyl-CoA oxidase, catalase, and PPAR-alpha(peroxisome-proliferator activated receptor-alpha)-dependent enzymes, such as malic enzyme and l-3-glycerophosphate dehydrogenase. NAD+ was also increased in the rat hepatocytes cultured in the medium supplemented with PPs. These data suggested that regulation of the key enzymes in the Trp-NAD+ pathway was associated with PPAR-alpha directly or indirectly, and as a consequence the hepatic NAD+ was increased by PPs., (Copyright 1999 Academic Press.)

Glutathion (GSH) was covalently attached to dextrans with various molecular weights of 2, 5, 10, 40, and 70 kDa by the cyanogen bromide activation method. The conjugates obtained synthetically were white or pale yellowish powders containing 6-10% (w/w) of GSH. The average molecular weights of the conjugates were estimated to be larger and the molecular weight distribution was a little broader than that of each original dextran. The conjugates significantly stabilized GSH and liberated it gradually under physiological conditions (t1/2 = 0.99-1.6h). Mice depleted of GSH by treatment with buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, exhibited a significant increase in hepatic GSH level after intravenous injection of the conjugates. In mice given a hepatotoxic dose of acetaminophen, the survival rate increased progressively with coadministration of the conjugates, whereas a small improvement was found when free GSH was given. The conjugate of GSH attached to dextran with the molecular weight of 40 kDa exhibited the highest prophylactic effect on acetaminophen-induced hepatotoxicity in mice. The prolonged retention of the conjugates of larger molecular weight in the circulation would cause a higher hepatic accumulation. These results suggested that molecular size would be the most critical factor in the delivery of GSH, as a dextran conjugate, into the liver.

Desialylated fetuin (asialofetuin) was adopted as a carrier for introducing drugs in parenchymal liver cells. Mitomycin C, as a model guest-compound, was covalently attached to asialofetuin through a spacer of the succinyl group. The asialofetuin-mitomycin C conjugate contained 4.4 w/w% of mitomycin C and liberated it gradually at physiological conditions (t1/2 = 37 h). The survival time of the conjugate in rat blood circulation was significantly smaller than that of the non-desialylated fetuin conjugate; the elimination half-life was 7.3 min after intravenous injection. At 30 min after injection of the conjugate in rats, 40% of the dose was present in the liver. Parenchymal cells in the liver selectively took up the conjugate, which was highly distributed to the lysosomal fraction in the cell. The greater uptake of the conjugate by hepatocytes reflected the increased excretion in the bile; totally 10.4% of the dose was recovered.

The effect of phenobarbital (PB) and 3-methylcholanthrene (3-MC) on the metabolic behavior of aminopyrine (AM) was studied using an isolated hepatocyte system prepared from male Wistar rats. The formation of 4-formylaminoantipyrine (FAA) was increased after pretreatment with PB, but not 3-MC. However, the total amounts of AM and its main metabolites recovered from hepatocyte system after the incubation for 30 min were considerably reduced both by PB and 3-MC-pretreatment. Furthermore, when using 4-monomethylaminoantipyrine (MAA), the first metabolite of AM, as a substrate, 3-MC-pretreatment resulted in a more significant decrease in the total amounts of MAA and its further metabolites recovered than did PB. Present observation suggests the participation of cytochrome P-448 as well as cytochrome P-450 in the metabolism of AM.

Glutathione was covalently attached to dextran (T-40) by the CNBr activation method. The compound obtained was a water-soluble powder containing 10 (w/w%) glutathione, which was gradually released from the conjugate in aqueous media. Mice depleted of glutathione by treatment with buthionine sulfoximine, a potent inhibitor of gamma-glutamylcysteine synthetase, exhibited a significant increase in hepatic glutathione level after intravenous injection of the conjugate. In mice given a lethal dose of acetaminophen, the survival rate increased progressively with coadministration of the conjugate, whereas little improvement was found when free glutathione was given. The conjugate maintained the serum transaminase activities at lower level after acetaminophen administration. These findings suggest that the dextran conjugate of glutathione is transported into hepatic cells and is intracellularly hydrolyzed to free form, which protects mice from hepatotoxicity due to acetaminophen.

Isoniazid metabolism in isolated rat hepatocytes was studied by mass fragmentography using single ion monitoring. Isoniazid and its metabolites were determined as the trimethylsilylated derivatives of acetylisoniazid and diacetylhydrazine and of the benzaldehyde hydrazones of isoniazid and acetylhydrazine. Deuterated analogues served as internal standards. Hydrazine was quantitated as benzalazine using 15N-labeled hydrazine as an internal standard. The method is well suited for the microanalysis of isoniazid metabolism, from which it was clarified that the greater part of hydrazine, a hazardous metabolite of isoniazid, was formed through the direct hydrolysis of isoniazid itself as expected.

Isoniazid (INH) was incubated with isolated rat hepatocytes, and the amounts of acetyl-INH, monoacetylhydrazine, diacetylhydrazine and hydrazine formed were determined by gas chromatography-mass spectrometry (GC-MS). Subsequent analyses of kinetic parameters of metabolic pathways and enzymological study of rat liver homogenate revealed that hydrazine was formed preferentially from INH, catalyzed by soluble hydrolytic enzymes.

The product inhibition of aminopyrine (AM) N-demethylation by 4-monomethylaminoantipyrine (MAA) was examined in vitro in preparations of isolated rat hepatocytes using gas chromatograph-mass spectrometer (GC-MS). The kinetic study for AM N-demethylation, which was determined by MAA formation, was studied at concentrations from 0.2 to 4.0 mM with or without various concentrations of D3-MAA. D3-MAA inhibited AM N-demethylation non-competitively at concentrations of 0.2 to 1.0 mM of AM (Ki = 0.81 mM), though it did competitively at concentrations of 1.0 to 4.0 mM of AM (Ki = 0.81 mM). From the present observation, it was suggested that the inhibition type of AM N-demethylation by D3-MAA was dependent on the substrate (AM) concentration.