Elimination of false-positive signals in enhanced chemiluminescence (ECL) detection of amplified HPV DNA from clinical samples.
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Cervix Uteri↗
Biomedical subjects
W Qu
Publications and source records attributed to W Qu.
Food restriction and stimulation of monooxygenation of p-nitroanisole in perfused rat liver.
This study assessed the effect of food restriction on the metabolism of model monooxygenase substrates in the perfused rat liver. Female Sprague-Dawley rats has access ad lib. to a Purina 5001 nonpurified diet (control) or were given 65% of the intake of controls for 3 weeks. Livers were perfused with oxygenated Krebs-Henseleit buffer using a non-recirculating system, and the rates of monooxygenation of p-nitroanisole and 7-ethoxycoumarin were measured. The results indicate that food restriction stimulated p-nitroanisole O-demethylation from 2.9 +/- 0.2 to 4.6 +/- 0.5 mumol/(g.hr) when saturating concentrations of p-nitroanisole were infused. Concomitantly, the ratio of beta-hydroxybutyrate to acetoacetate (B/A) and the rates of ketogenesis (B + A) were increased significantly by food restriction. Further, p-nitroanisole (200 mumol/L) increased hepatic malate concentration nearly 3-fold in liver extracts from food-restricted rats. However, infusion of either a low concentration of p-nitroanisole (50 mumol/L) or 7-ethoxycoumarin (200 mumol/L) did not alter these parameters. On the other hand, food restriction did not alter rates of monooxygenation in isolated microsomes supplemented with excess NADPH. Taken together, these data support the hypothesis that high concentrations of p-nitroanisole increased monooxygenation in food-restricted rats by stimulating fatty acid oxidation, which elevates the mitochondrial NADH/NAD+ ratio. This, in turn, increases the availability of reducing equivalents in the form of NADPH by a malate-pyruvate exchange system, leading to increased drug metabolism.
7-Alkoxycoumarin O-Dealkylase↗
Increase in oxygen uptake due to arachidonic acid is oxygen dependent in the perfused liver.
The purpose of this study was to determine whether the effect of arachidonic acid on hepatic O2 uptake is O2 dependent and which region of the liver lobule it affects. In livers perfused at normal flow rates, infusion of arachidonate increased O2 uptake significantly by about 20-25 mumol.g-1.h-1. When the flow rate was doubled to make the hepatic O2 gradient shallower, the increase in O2 uptake due to arachidonate was two to three times larger (i.e., approximately 50 mumol.g-1.h-1). In livers perfused in the retrograde direction, maximal rates of O2 uptake were about twofold higher in upstream pericentral than in downstream periportal regions, and arachidonic acid nearly doubled O2 uptake in downstream areas without affecting rates in upstream regions. Thus it is concluded that arachidonate stimulates O2 uptake in an O2-dependent manner. This effect was sensitive to an inhibitor of the lipoxygenase, nordihydroguaiaretic acid, in perfused liver but not in isolated hepatocytes. In addition, conditioned medium from Kupffer cells incubated at high O2 tension stimulated parenchymal cell O2 uptake. Furthermore, arachidonate increased intracellular Ca2+ in isolated Kupffer cells in a dose-dependent manner. These findings suggest that eicosanoids produced by nonparenchymal cells participate in a hepatic O2 sensor mechanism involving Ca2+ that regulates O2 uptake by parenchymal cells in the liver.
Animals↗
Food restriction increases detoxification of polycyclic aromatic hydrocarbons in the rat.
It is well known that food restriction diminishes tumor formation, but mechanisms responsible are difficult to define because multiple physiological changes result from dietary alterations. Studies in this report were designed to focus specifically on the effects of food restriction on hepatic metabolism of polycyclic aromatic hydrocarbons following liver transplantation. By placing livers from food-restricted and untreated rats into naive controls, the effects of diet could be restricted to the liver. After a 15 min infusion of [3H]benzo[a]pyrene under these conditions, food restriction increased polar metabolites in liver (70 pmol/g) and blood (8 pmol/ml) compared to controls approximately 2-fold. Four hours after liver transplantation, levels of polar metabolites in blood were diminished by approximately 50% but were still approximately 2-fold higher in the food-restricted than in the control group. Lung, kidney, spleen, adrenal, ovary, colon, heart and brain also contained higher levels of polar metabolites in the food-restricted than in the control group. The more hydrophobic glucuronides and sulfate conjugates accounted for most of the elevation in polar metabolites in blood from the food-restricted group. In spite of the increase in circulating metabolites in blood of food-restricted animals, DNA binding in liver, lung and kidney was identical in tissues from control and food-restricted groups. In order to evaluate the hypothesis that food restriction stimulated the release of hepatic benzo[a]pyrene metabolites, a liver perfusion model was employed. Maximal rates of release of polar metabolites into the effluent perfusate were approximately 30 and approximately 45 nmol/g/h in livers of control and food-restricted rats respectively. Moreover, rates of metabolism of the model compound p-nitroanisole and glucuronidation of p-nitrophenol were also approximately 2-fold higher in livers from food-restricted than control rats. However, rates of monooxygenation were the same in microsomes prepared from livers of food-restricted or control animals. These results support the hypothesis that food restriction enhances the supply of cofactors which stimulate metabolism of polycyclic aromatic hydrocarbons. This detoxification process may be an important mechanism involved in the protective action of reduced food intake.
Animals↗
Stimulation of monooxygenation and conjugation after liver transplantation in the rat: involvement of Kupffer cells.
The success rate of liver transplantation has improved markedly during the last few years and, although this patient population receives multiple drug therapies, the effect of liver transplantation on drug metabolism has been studied very little. Therefore, the purpose of this study was to assess the metabolism of model drug substrates after liver transplantation in the rat. Rat livers were stored for 4 hr in cold Euro-Collins solution, transplanted orthotopically, and then perfused 2 hr later with oxygenated Krebs-Henseleit buffer, using a nonrecirculating system. Rates of monooxygenation of the model compound p-nitroanisole, conjugation of p-nitrophenol, and uptake of oxygen were measured. All parameters studied were elevated significantly, by nearly 2-fold, by transplantation. Specifically, monooxygenation was increased from 2.9 +/- 0.2 to 5.1 +/- 0.4 mumol/g/hr, conjugation was elevated from 3.3 +/- 0.6 to 7.7 +/- 0.1 mumol/g/hr, and O2 uptake was stimulated from basal values of 114 to 197 mumol/g/hr. Transplantation did not, however, alter rates of monooxygenation and conjugation in isolated microsomes supplemented with excess cofactor. When donor rats were pretreated with the Kupffer cell toxicant gadolinium chloride (10 mg/kg, intravenously) 30 hr before liver storage, the elevation after transplantation in all parameters studied was prevented. Depletion of carbohydrate reserves by fasting of donor rats did not prevent stimulation of monooxygenation and conjugation. On the other hand, urea synthesis from ammonium chloride, a process dependent on mitochondrial NADPH, was increased and monooxygenation was diminished after transplantation, suggesting the involvement of mitochondria in this phenomenon. Indeed, mitochondria isolated 2 hr postoperatively exhibited significantly elevated respiratory control ratios and higher state 3 rates of respiration. Taken together, these data support the hypothesis that Kupffer cells, activated by transplantation, release mediators that stimulate mitochondria in parenchymal cells and enhance drug metabolism by increasing cofactor supply (e.g., NADPH for monooxygenation and UDP-glucuronic acid for glucuronidation).
Ammonium Chloride↗
Unique role of oxygen in regulation of hepatic monooxygenation and glucuronidation.
The purpose of this study was to evaluate the hypothesis that NADPH supply in intact cells is regulated by oxygen tension. This was accomplished by studying monooxygenation in perfused livers from Ah locus-responsive C57BL/6J mice, where rates of monooxygenation are high. Elevation of flow rate decreases the hepatic O2 gradient and increases O2 delivery to the organ. Under these conditions, rates of p-nitroanisole O-demethylation were 2-3 times higher in perfused livers from fed or fasted mice at high (10 ml/min) compared with normal (5 ml/min) flow rates. Rates of monooxygenation were directly proportional to oxygen tension (half-maximal rates occurred with approximately 400 microM O2). On the other hand, rates were independent of oxygen concentration in isolated microsomes where NADPH was supplied in excess. The decrease in rate due to diminished O2 concentration in the intact organ could not be attributed to hypoxia, because O2 tension in the effluent perfusate exceeded 50 microM even when influent perfusate was saturated with 25% O2 and ATP/ADP ratios were in the normal range. Thus, monooxygenation of p-nitroanisole in perfused mouse liver is dependent on oxygen tension. Similarly, glucuronidation of p-nitrophenol was oxygen dependent in the intact organ but not in isolated microsomes supplemented with UDP-glucuronic acid. Taken together, these data support the hypothesis that, at high oxygen tensions (e.g., in periportal regions of the liver lobule), mitochondrial activity is increased, which in turn enhances NADPH and UDP-glucuronic acid turnover, leading to accelerated rates of monooxygenation and glucuronidation in intact cells. In support of this idea, NH4Cl, which utilizes NADPH for urea synthesis, inhibited monooxygenation in the perfused mouse liver at high but not low flow rates. Thus, important phase I and II detoxification reactions are regulated indirectly by the hepatic oxygen gradient, via mechanisms involving cofactor supply, when cytochrome P-450 is not limiting.
Animals↗