Steven A. Belinsky

Co-regulation of the mixed-function oxidation of p-nitroanisole and glucuronidation of p-nitrophenol in the perfused rat liver by carbohydrate reserves.

Maximal rates of mixed-function oxidation of p-nitroanisole and the glucuronidation of p-nitrophenol in perfused livers from phenobarbital-treated rats varied directly with the nutritional state of the rat (i.e., fasted < fed < fasted-refed). Rates correlated with intracellular concentrations of NADPH, UDP-glucuronic acid, and glycogen but not with amounts of cytochrome P-450 or glucuronyltransferase activity. These data support the hypothesis that mixed-function oxidation and glucuronidation are coregulated in intact cells by carbohydrate-dependent cofactor synthesis.

Regulation of NADPH-dependent mixed-function oxidation in perfused livers: Comparative studies with sorbitol and ethanol☆

Sorbitol and ethanol were shown to have opposite effects on p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Sorbitol (2 mM) stimulated drug metabolism by 50% while ethanol (20 mM) caused 80% inhibition. Both sorbitol and ethanol infusion decreased the NAD+/NADH ratio and increased fluorescence of pyridine nucleotides monitored from the liver surface; however, the NADP+/NADPH ratio was decreased by sorbitol but tended to be increased by ethanol. Stimulation of drug metabolism by sorbitol was abolished by pretreatment of fasted rats with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt, but was not affected by aminooxyacetate, a transaminase inhibitor. These results indicate that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. Ethanol and sorbitol caused changes in intracellular concentrations of NADPH in livers from fasted rats which correlated directly with changes in hepatic levels of citrate and aspartate. Furthermore, aspartate infusion reduced the inhibition of p-nitroanisole O-demethylation by ethanol. This inhibition was also reversed partially by sorbitol in livers from 6-aminonicotinamide-treated rats. It is concluded that ethanol inhibits mixed-function oxidation primarily by decreasing the concentrations of citric acid cycle intermediates which leads to depletion of cytosolic NADPH.

Inhibition of mixed-function oxidation of p-nitroanisole in perfused rat liver by 2,4-dinitrophenol☆

Abstract The effect of dinitrophenol (52 μ m ), an uncoupler of oxidative phosphorylation, on p-nitroanisole O-demethylation in the perfused rat liver was examined. Dinitrophenol inhibited p-nitroanisole metabolism 70% in perfused livers from fasted, phenobarbital-treated rats, and 30% in livers from normal rats, but had no effect on this reaction in isolated microsomes. Rates of p-nitroanisole O-demethylation in livers from fed, phenobarbitaltreated rats were not inhibited by dinitrophenol unless the pentose phosphate shunt was first inhibited by 6-aminonicotinamide pretreatment. Dinitrophenol diminished cellular concentrations of ATP and NADPH 30 and 50%, respectively. Since mixed-function oxidation requires NADPH, these data are consistent with the hypothesis that dinitrophenol interrupts the synthesis and/or transfer of reducing equivalents from the mitochondria into the extramitochondrial space by interfering with energy-dependent NADPH synthesis and substrate shuttle mechanisms. In addition, dinitrophenol diminished conjugation reactions 57 and 89% in all metabolic states studied, most likely because it decreased UDP-glucose levels considerably (40 to 60%).

Inhibition of p-nitroanisole O-demethylation in perfused rat liver by potassium cyanide.

The effect of potassium cyanide on p-nitroanisole O-demethylation in perfused rat livers has been examined. Cyanide (2 mM), an inhibitor of cytochrome oxidase, diminished p-nitroanisole O-demethylation by 50-75% in perfused livers from normal and phenobarbital-treated rats, but had much less effect on hepatic microsomal p-nitroanisole O-demethylation. The inhibition was also observed in livers where the activity of the pentose phosphate shunt was abolished by pretreatment with 6-aminonicotinamide. Cyanide infusion decreased hepatic ATP/ADP ratios and cellular concentrations of glutamate, alpha-ketoglutarate, and isocitrate, but caused an increase in the NADP+/NADPH ratio. Rates of NADPH generation via the pentose phosphate shunt were unchanged by cyanide, and hepatic concentrations of glucose 6-phosphate were markedly increased by cyanide. Thus, inhibition of p-nitroanisole metabolism could not be explained solely by a direct interaction of cyanide with mixed-function oxidases or diminished NADPH generation via the pentose cycle. These data indicate that cyanide inhibits mixed-function oxidation in intact cells by diminishing the generation of NADPH from sources other than the pentose cycle. Further, these data are consistent with the hypothesis that some NADPH for mixed-function oxidation arises from cyanide-sensitive mitochondrial sources.

p-Nitroanisole O-demethylation in perfused hamster liver: High rates of pentose cycle-independent mixed-function oxidation☆

Rates of p-nitroanisole O-demethylation in perfused livers from Syrian golden hamsters were three to four times greater than comparable rates measured in preparations from Sprague-Dawley rats. Hamsters also had greater microsomal p-nitroanisole O-demethylase activity and cytochrome P-450 contents than rats. In general, phenobarbital caused similar increases in these properties in both species. Fasting of hamsters for 24 hr increased p-nitroanisole O-demethylase activity in microsomes but did not affect rates in perfused livers. Rates were also unaffected in the perfused liver by pretreatment with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt. Hamster livers had low activities of pentose cycle enzymes but high activities of malic enzyme and isocitrate dehydrogenase compared to rats. In hamster livers, maximal rates of p-nitroanisole O-demethylation were not maintained but declined steadily over 40 min with prolonged p-nitroanisole infusion. The decreased rates of mixed-function oxidation in the non-recirculating perfusion system could not be explained by diminished tissue viability or degradation of cytochrome P-450 but were likely due to a decline in the formation of reduced cofactor. Hepatic concentrations of alpha-ketoglutarate and malate increased during p-nitroanisole infusion. Furthermore, rates of p-nitroanisole O-demethylation were inhibited by ethanol and aminooxyacetate, agents which inhibit the generation and/or movement of mitochondrial reducing equivalents into the cytosol. The infusion of pyruvate stimulated p-nitroanisole O-demethylation in perfused livers from fasted hamsters. This effect was maximal with 0.1 mM pyruvate, did not require gluconeogenesis, and was insensitive to 6-aminonicotinamide treatment. Thus, stimulation of p-nitroanisole metabolism by pyruvate in hamster livers is likely related to the mitochondrial oxidation of pyruvate, rather than to increased NADPH generation via the pentose phosphate cycle. These data indicate that mitochondrial sources of NADPH supply reducing equivalents for mixed-function oxidation in hamster liver.

Effect of allyl alcohol on xanthine dehydrogenase activity in the perfused rat liver

Xanthine oxidase has been implicated in the production of reactive oxygen species and cell injury produced by various toxic compounds. Since allyl alcohol injuries the liver by an oxygen-dependent mechanism, we examined the actions of this hepatotoxicant on the conversion of xanthine dehydrogenase into xanthine oxidase in perfused livers. A microassay for NAD(+)-dependent xanthine dehydrogenase, based on measuring the production of NADH fluorometrically under anaerobic conditions, was developed and used to examine the actions of allyl alcohol on this activity in periportal and pericentral regions of the liver lobule. The oxygen-dependent activity, xanthine oxidase, was monitored in whole liver homogenates by uric acid formation at 302 nm under aerobic conditions. Perfusion of the liver with allyl alcohol (350 microM) increased xanthine oxidase and decreased xanthine dehydrogenase in whole liver consistent with the hypothesis that allyl alcohol enhanced calcium-dependent proteolytic conversion of the NAD(+)-dependent to the O2-dependent form. Xanthine dehydrogenase was higher in pericentral than in periportal regions of the liver lobule and tended to decrease selectively in periportal zones of livers exposed to allyl alcohol. O2 uptake was stimulated transiently by allyl alcohol followed by subsequent inhibition of respiration. These results are consistent with the idea that conversion of NAD(+)-dependent xanthine dehydrogenase to xanthine oxidase is involved in the zone-specific hepatotoxicity of allyl alcohol.

Metabolic heterogeneity in the perfused rat liver

New methods have been developed to monitor metabolic events non-invasively within periportal and pericentral regions of perfused rat livers. These techniques utilize two-fiber micro-light guides and miniature oxygen electrodes positioned on identified lobular regions of the perfused liver based on differential pigmentation of periportal and pericentral areas. Two-fiber micro-light guides detect the fluorescence of native and introduced fluors and are used to monitor redox changes of endogenous pyridine nucleotides and the generation of fluorescent products (e.g., 7-hydroxycoumarin) from exogenous substrates. Changes in fluorescence detected with two-fiber micro-light guides are correlated with changes measured with large, multi-fiber light guides and with whole organ rates of metabolism. Subsequently, local rates are estimated. With these techniques, we show that (a) rates of ethanol and acetaldehyde metabolism are similar in periportal and pericentral regions of the liver lobule; (b) mixed-function oxidation predominantes in pericentral regions in livers from phenobarbital-treated rats; (c) rates of sulfation of 7-hydroxycoumarin are greater in periportal than in pericentral hepatocytes; and (d) oxygen uptake is approximately 3-fold greater in periportal than in pericentral areas of the liver lobule.

Effects of ethanol and sorbitol on mixed-function oxidation in perfused rat livers ☆

Ethanol (20 mM) caused 50-90% inhibition of rates of mixed-function oxidation of p-nitroanisole, 7-ethoxycoumarin and benzo(a)pyrene in perfused rat livers; however, the microsomal metabolism of these substrates was unaltered by low concentrations of ethanol. The metabolism of ethanol was required for this inhibition in the perfused liver. In contrast to ethanol, sorbitol stimulated rates of p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Both sorbitol and ethanol infusion decreased the hepatic NAD+/NADH ratio; however, the NADP+/NADPH ratio was decreased by sorbitol but increased by ethanol. Stimulation of drug metabolism by sorbitol was abolished by pretreatment of fasted rats with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt. These data indicated that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. The changes in intracellular concentrations of NADPH produced by ethanol and sorbitol correlated directly with changes in hepatic content of citrate and aspartate. These data suggest that inhibition of the citric acid cycle by ethanol decreases the movement of mitochondrial reducing equivalents into the cytosol via substrate shuttle mechanisms.

Mechanism of inhibition of mixed-function oxidation by ethanol.

p-Nitroanisole (PNA) O-demethylation in perfused rat livers was inhibited by low concentrations of ethanol (Ki = 1 mM) and acetaldehyde (Ki = 0.5 mM). Ethanol and acetaldehyde also inhibited microsomal PNA metabolism, but only at much higher concentrations (Ki = 130 mM and 5 mM, respectively). Moreover, the dose-dependent inhibition of PNA metabolism in the perfused liver correlated strongly with changes in surface fluorescence of pyridine nucleotides and flavoproteins resulting from the metabolism of ethanol and acetaldehyde. t-Butanol (20 mM) did not cause pyridine nucleotide or flavoprotein reduction, nor did it inhibit PNA metabolism. When ethanol metabolism was blocked with 4-methylpyrazole, the inhibition of PNA metabolism by ethanol was diminished.

ROLE OF MITOCHONDRIA IN ZONE-SPECIFIC TOXICITY TO THE LIVER LOBULE

Recently developed non-invasive optical and polarographic techniques along with ultramicrobiochemical methods were used to study the actions of allyl alcohol, a classic periportal toxin, on biochemical events in sublobular zones of the isolated perfused liver. Anterograde infusion of allyl alcohol (350 μM) for 60 minutes selectively injured periportal hepatocytes as indexed by uptake of trypan blue and by decreases in ATP/ADP ratios. Zone-specific decreases in ATP/ADP ratios were accompanied by impaired rates of oxygen uptake. Potassium cyanide (2 mM), an inhibitor of cytochrome oxidase, decreased ATP/ADP ratios but did not increase trypan blue uptake. Thus, disruption of ATP generation is a consequence rather than a cause of cellular injury due to allyl alcohol. Rates of oxidation of allyl alcohol to acrolein via alcohol dehydrogenase and depletion of cellular glutathione occurred equally in both zones of livers exposed to allyl alcohol indicating that zonal toxicity due to allyl alcohol was not due to differences in rates of metabolism of this compound.