Cytochrome P450 2E1-derived reactive oxygen species mediate paracrine stimulation of collagen I protein synthesis by hepatic stellate cells.

To evaluate possible fibrogenic effects of CYP2E1-dependent generation of reactive oxygen species, a model was developed using co-cultures of HepG2 cells, which do (E47 cells) or do not (C34 cells) express cytochrome P450 2E1 (CYP2E1) with stellate cells. There was an increase in intra- and extracellular H(2)O(2), lipid peroxidation, and collagen type I protein in stellate cells co-cultured with E47 cells compared with stellate cells alone or co-cultured with C34 cells. The increase in collagen was prevented by antioxidants and a CYP2E1 inhibitor. CYP3A4 did not mimic the stimulatory effects found with CYP2E1. Collagen mRNA levels remained unchanged, and pulse-chase analysis indicated similar half-lives of collagen I protein between both co-cultures. However, collagen protein synthesis was increased in E47 co-culture. Hepatocytes from pyrazole-treated rats (with high levels of CYP2E1) induced collagen protein in primary stellate cells, and antioxidants and CYP2E1 inhibitors blocked this effect. These results suggest that increased translation of collagen mRNA by CYP2E1-derived reactive oxygen species is responsible for the increase in collagen protein produced by the E47 co-culture. These co-culture models may be useful for understanding the impact of CYP2E1-derived ROS on stellate cell function and activation.     

The effect of acetaldehyde on gluconeogenesis from xylitol, sorbitol, and fructose by isolated rat liver cells.

In isolated rat liver cells, ethanol inhibited gluconeogenesis from xylitol and sorbitol but not from fructose. Acetaldehyde, at initial concentrations of 0.2, 0.5, and 1.0 mm, stimulated gluconeogenesis from xylitol and sorbitol in the absence of pyrazole but inhibited in the presence of pyrazole. There was no effect with fructose. Acetate had no effect. Methylene blue and pyruvate (but not lactate) prevented the stimulatory as well as the inhibitory effects of acetaldehyde. Acetoacetate (but not β3-hydroxybutyrate) prevented, to a large extent, the inhibitory effects of low (but not high) concentrations of acetaldehyde. The inhibition by low concentrations of acetaldehyde appears to be mediated via acetaldehyde oxidation in the mitochondria, whereas the inhibition by high concentrations of acetaldehyde appears to reflect acetaldehyde oxidation in the cytosol. These data indicate that the inhibitory action of ethanol on glucose production from xylitol and sorbitol can be reproduced by physiological concentrations of acetaldehyde. Changes in the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio produced during acetaldehyde metabolism appear to be responsible for these effects of acetaldehyde. These changes may contribute to the actions of ethanol on gluconeogenesis from these substrates.     

Production of 4-hydroxypyrazole from the interaction of the alcohol dehydrogenase inhibitor pyrazole with hydroxyl radical

Pyrazole, an effective inhibitor of alcohol dehydrogenase, was previously shown to be a scavenger of the hydroxyl radical. 4-Hydroxypyrazole is a major metabolite in the urine of animals administered pyrazole in vivo. Experiments were conducted to show that 4-hydroxypyrazole was a product of the interaction of pyrazole with hydroxyl radical generated from three different systems. The systems utilized were the iron-catalyzed oxidation of ascorbate, the coupled oxidation of hypoxanthine by xanthine oxidase, and NADPH-dependent microsomal electron transfer. Ferric-EDTA was added to all the systems to catalyze the production of hydroxyl radicals. A HPLC procedure employing either uv detection or electrochemical detection was utilized to assay for the production of 4-hydroxypyrazole. The three systems all supported the oxidation of pyrazole to 4-hydroxypyrazole by a reaction which was sensitive to inhibition by competitive hydroxyl radical scavengers such as ethanol, mannitol, or dimethyl sulfoxide and to catalase. The sensitivity to catalase implicates H2O2 as the precursor of the hydroxyl radical by all three systems. Superoxide dismutase inhibited production of 4-hydroxypyrazole only in the xanthine oxidase reaction system. In the absence of ferric-EDTA (and azide), microsomes catalyzed the oxidation of pyrazole to 4-hydroxypyrazole by a cytochrome P-450-dependent reaction which was independent of hydroxyl radicals. This latter pathway may be primarily responsible for the in vivo metabolism of pyrazole to 4-hydroxypyrazole. The production of 4-hydroxypyrazole from the interaction of pyrazole with hydroxyl radicals may be a sensitive, rapid technique for the detection of these radicals in certain tissues or under certain conditions, e.g., increasing oxidative stress.     

Interaction of ferric complexes with rat liver nuclei to catalyze NADH-and NADPH-Dependent production of oxygen radicals

The production of potent oxygen radicals by microsomal reaction systems has been well characterized. Relatively little attention has been paid to generation of oxygen radicals by liver nuclei, or to the interaction of nuclei with different ferric complexes to catalyze NADH- or NADPH-dependent production of reactive oxygen intermediates. Intact rat liver nuclei were capable of catalyzing an iron-dependent production of .OH as reflected by the oxidation of .OH scavenging agents such as 2-keto-4-thiomethylbutyrate, dimethyl sulfoxide, and t-butyl alcohol. Inhibition of .OH production by catalase implicates H2O2 as the precursor of .OH generated by the nuclei, whereas superoxide dismutase had only a partially inhibitory effect. The production of .OH with either cofactor was striking increased by addition of ferric-EDTA or ferric-diethylenetriamine-pentaacetic acid (DTPA) whereas ferric-ATP and ferric-citrate were not effective catalysts. All these ferric complexes were reduced by the nuclei in the presence of either NADPH or NADH. The pattern of iron chelate effectiveness in catalyzing lipid peroxidation by nuclei was opposite to that of .OH production; with either NADH or NADPH, nuclear lipid peroxidation was increased by the addition of ferric ammonium sulfate, ferric-ATP, or ferric-citrate, but not by ferric-EDTA or ferric-DTPA. NADPH-dependent nuclear lipid peroxidation was insensitive to catalase, superoxide dismutase, or .OH scavengers; the NADH-dependent reaction showed a partial sensitivity (30 to 40%) to these additions. The overall patterns of .OH production and lipid peroxidation by the nuclei are similar to those shown by microsomes, e.g., effect of ferric complexes, sensitivity to antioxidants; however, rates with the nuclei are less than 20% those of microsomes, which reflect the lower activities of NADPH- and NADH-cytochrome c reductase in the nuclei. The potential for nuclei to reduce ferric complexes and catalyze production of .OH-like species may play a role in the susceptibility of the genetic material to oxidative damage under certain conditions since such radicals would be produced site-directed and not exposed to cellular antioxidants.     

Increased NADPH-dependent chemiluminescence by microsomes after chronic ethanol consumption

The generation of reactive oxygen intermediates by microsomes from ethanol-fed rats and pair-fed controls was determined by assaying for NADPH-dependent chemiluminescence. In the absence or presence of added ferric complexes, microsomal light emission was elevated several-fold after chronic ethanol consumption. Iron complexes such as ferric-citrate or ferric-ATP stimulated, while ferric-EDTA, inhibited microsomal chemiluminescence. Freeze-thawing the microsomes to elevate their content of lipid hydroperoxides resulted in large increases in chemiluminescence; under all conditions, the light emission remained several-fold higher with microsomes from the ethanol-fed rats. Chemiluminescence was not sensitive to superoxide dismutase, catalase, or the hydroxyl radical scavenging agent, dimethyl sulfoxide, but was inhibited by antioxidants and by glutathione. Replacing air with a mixture of 50% nitrogen-50% air or 50% carbon monoxide-50% air had no effect on chemiluminescence by microsomes from the pair-fed controls. However, the chemiluminescent response by microsomes from the ethanol-fed rats was inhibited about 50% by the nitrogen mixture, and was further inhibited (about 75% of values found with 100% air, and 50% of values found with 50% nitrogen-50% air) with the carbon monoxide mixture. The sensitivity to carbon monoxide suggests the possibility that the alcohol-inducible cytochrome P-450 isozyme may contribute, in part, to the elevated light emission produced by microsomes from the ethanol-fed rats. The increase in chemiluminescence by microsomes after chronic ethanol consumption appears to reflect an elevated level of lipid hydroperoxides as well as an increased rate of generation of reactive oxygen species.     

Increased microsomal interaction with iron and oxygen radical generation after chronic acetone treatment

In vivo administration of acetone influences a variety of reactions catalyzed by rat liver microsomes. The effect of chronic treatment with acetone (1% acetone in the water for 10-12 days) on interaction with iron and subsequent oxygen radical generation by liver microsomes was evaluated. Microsomes from the acetone-treated rats displayed elevated rates of H2O2 generation, an increase in iron-dependent lipid peroxidation, and enhanced chemiluminescence upon the addition of t-butylhydroperoxide. The ferric EDTA-catalyzed production of formaldehyde from DMSO or of ethylene from 2-keto-4-thiomethylbutyrate was increased 2-fold after acetone treatment. This increase in hydroxyl radical generation was accompanied by a corresponding increase in NADPH utilization and was sensitive to inhibition by catalase and a competitive scavenger, ethanol, but not to superoxide dismutase. In vitro addition of acetone to microsomes had no effect on oxygen radical generation. Associated with the chronic acetone treatment was a 2-fold increase in the microsomal content of cytochrome P-450 and in the activity of NADPH-cytochrome-P-450 reductase. It appears that increased oxygen radical generation by microsomes after chronic acetone treatment reflects the increase in the major enzyme components which comprise the mixed-function oxidase system.     

Comparison of arginase activity in red blood cells of lower mammals, primates, and man: evolution to high activity in primates.

Arginase activity in red blood cells (RBC) of various mammalian species including man was determined. In nonprimate species, the activity generally fell below the level of detectability of the assay: less than 1.0 mumol urea/g hemoglobin per hr. Activities in higher nonhuman primates were equal to or of the same order of magnitude as those in man (approximately 950 mumol/g hemoglobin per hr). RBC arginase deficiency with normal liver arginase activity has been shown to segregate as an autosomal codominant trait in Macaca fascicularis established and bred in captivity. This study confirms the presence of this polymorphism in wild populations trapped in several geographic areas and demonstrates the absence of immunologically cross-reactive material in the RBC of RBC arginase-deficient animals. These data when taken together suggest that the expression of arginase in RBC is the result of a regulatory alteration, has evolved under positive selective pressure, and is not an example of the vestigial persistence of an arcane function. The expression of arginase in the RBC results in a marked drop in the arginine content of these cells.     

Inhibition of microsomal oxidation of ethanol by pyrazole and 4-methylpyrazole in vitro. Increased effectiveness after induction by pyrazole and 4-methylpyrazole.

[2-18O]Ribulose 5-phosphate was prepared and shown to be converted enzymically by 6-phosphogluconate dehydrogenase from sheep liver into 6-phosphogluconate with complete retention of the heavy isotope. This finding unequivocally excludes the possibility of a Schiff-base mechanism for the enzyme. The involvement of metal ions has already been excluded, and other possible mechanisms are discussed. The enzyme was purified by an improved large-scale procedure, which is briefly described.     

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.