Canalicular transport of reduced glutathione in normal and mutant Eisai hyperbilirubinemic rats.

We have characterized the transport of GSH and the mechanism for impaired GSH transport in mutant Eisai hyperbilirubinemic rats (EHBR) using isolated canalicular membrane-enriched vesicles (cLPM). In control animals, the transport of GSH is an electrogenic process and is trans-stimulated by preloading the vesicles with GSH and is not enhanced in the presence of ATP. GSH transport in cLPM is saturable with a single component having a Km of approximately 16 mM and a Vmax of 6.7 nmol/mg/15 s. EHBR is a Sprague-Dawley rat with hyperbilirubinemia due to impaired bile secretion of organic anions by the ATP-dependent organic anion/GSH-conjugate transporter. In cLPM from EHBR we confirmed the defective stimulation by ATP of the transport of LTC4 and GSSG. In the mutant cLPM, the characteristics and kinetics of GSH transport were the same as in the controls. 2,4-(dinitrophenyl)-glutathione (DNP-GSH), which is a substrate for the ATP-dependent canalicular organic anion carrier, in the absence of ATP, cis-inhibited the transport of GSH into cLPM vesicles; however, when the vesicles were preloaded with DNP-GSH, there was a dose-dependent trans-stimulation of GSH transport. In contrast, in the presence of ATP, DNP-GSH enhanced GSH transport in cLPM vesicles; at 0.25 mM DNP-GSH, a concentration which did not cis-inhibit GSH, addition of ATP resulted in accelerated GSH transport; at 1.0 mM DNP-GSH, cis-inhibition was completely reversed by the addition of ATP despite a negligible fall in the medium DNP-GSH. Interestingly, sulfobromophthalein-glutathione (BSP-GSH) neither cis-inhibited nor trans-stimulated GSH transport in cLPM. This contrasts with bLPM where BSP-GSH interacts with the GSH carrier. Therefore, GSH is transported into bile by a multispecific low affinity electrogenic carrier which is distinct from the multispecific high affinity ATP-driven organic anion transporter. Although both carriers have overlapping specificities, BSP-GSH and GSH are uniquely specific for only one of the carriers. The near absence of GSH in the bile of mutant rats can be best explained as a secondary defect due to cis-inhibition from retained substrates for the defective carrier and/or loss of trans-stimulation by these same substrates which normally are concentratively transported into the bile. Other possibilities such as change in GSH carrier activity upon isolation or loss of a negative protein regulator during membrane isolation, although theoretical alternatives are less easily reconciled with the defect in the ATP-driven organic anion transporter.     

Biliary excretion of copper in fischer rats treated with copper salt and in long-evans cinnamon (LEC) rats with an inherently abnormal copper metabolism

Increased biliary Cu excretion was found in Fischer rats injected with Cu. The biliary Cu was located at the void (large-molecule region) and total (small-molecule region) volume of a Sephadex G-75 column. The most Cu was found in the total volume. The two Cu peaks comigrated with absorbance at 280 nm. Although the bile from Cu-untreated Fischer rats did not show Cu absorbance in the total volume, absorbance at 280 nm was also found in this region. Even though Long-Evans Cinnamon (LEC) rats deposited a gross amount of Cu (194.0±27.8 μg/g liver) in the liver, they conversely showed reduced Cu excretion into the bile. LEC bile did not show Cu absorbance but rather absorbance at 280 nm in the total volume. Therefore, it seems unlikely that the small molecules found in the Sephadex G-75 regulate biliary Cu excretion in Cu-loaded rats, although the molecules bind to Cu. When the bile from Cu-untreated fischer and LEC rats was incubated with CuCl₂ solution, the most Cu was recovered in the total volume of this column. Our results suggest that reduced biliary Cu excretion in LEC rats is not related to the small molecules, and that Cu cannot be excreted in the form of macromolecules in rats to decrease Cu from the Cu-loaded liver.     

Biliary copper excretion capacity in intact animals: correlation between ATP7B function, hepatic mass, and biliary copper excretion

Copper toxicosis can occur in the absence of biliary copper excretion. To demonstrate whether biliary copper excretion capacity is correlated with hepatic mass and ATP7B function, we undertook studies in intact animals. Copper-histidine was injected intrasplenically after baseline bile collection, followed by measurement of copper excretion in Long-Evans Cinnamon (LEC) rats lacking atp7b function and in normal, syngeneic Long-Evans Agouti (LEA) rats. The basal biliary copper excretion was very low in LEC rats compared with LEA rats, 8+/-4 and 37+/-18 ng copper/min, respectively; p<0.05. After addition of copper, copper excretion increased significantly (by two- to five-fold) in LEA rats during the 30 minute study period, whereas LEC rats showed only a slight and transient increase in copper excretion. After one-third and two-thirds partial hepatectomy immediately before copper loading, copper excretion decreased progressively. The studies indicate that biliary copper excretion depends on hepatocyte mass and ATP7B gene function. Analysis of copper excretion with our non-radioactive method will facilitate testing of novel therapies and pathophysiological mechanisms in copper toxicity.     

Ascorbic acid feeding of rats reduces copper absorption,causing impaired copper status and depressed biliary copper excretion

The feeding of diets enriched with ascorbic acid (10 g/kg) to rats has previously been shown to lower plasma and liver copper concentrations. The present studies corroborate this. We hypothesized that ascorbic acid initially reduces copper absorption, this effect being masked later by the stimulatory effect on copper absorption of the impaired copper status. We also hypothesized that the impaired copper status as induced by ascorbic acid feeding is followed by a diminished biliary excretion of copper in an attempt to preserve copper homeostasis. Our hypotheses are supported by the present studies. Ascorbic acid feeding initially reduced apparent copper absorption, and in the course of the experiment this effect tended to turn over into a stimulatory effect. Copper deficiency, as induced by feeding a diet containing 1 mg Cu/kg instead of 5 mg Cu/kg, systematically increased copper absorption. Biliary excretion of copper in rats given ascorbic acid was unaffected initialy but became depressed after prolonged ascorbic acid feeding. A similar time course was seen for fecal endogenous copper excretion that was calculated as the difference between true and apparent copper absorption. Copper deficiency systematically reduced biliary copper excretion and fecal endogenous copper loss.     

Biliary excretion of metallothionein and a possible degradation product in rats injected with copper and zinc.

The concentrations of metallothionein-I (MT-I) and related immunoreactive products in bile from adult female rats were measured by radioimmunoassay. Concentrations in normal animals were 20-30 ng/ml, but increased to 600 and 75 ng/ml after injection of Cu2+ and Zn2+ respectively (3 mg of metal/kg body wt.). However, only 1-2% of the biliary Cu was bound to MT, and less than 1% of the total liver MT in control or Cu2+-injected rats appeared to be secreted in intact form into bile. Other major immunoreactive components in bile from Cu2+-injected rats included an aggregated form of MT-I and a possible degradation product of the isoprotein.     

Vanadium effect on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase in vitro.

The effect of vanadium (V) on the activity of horseradish peroxidase, catalase, glutathione peroxidase, and superoxide dismutase has been studied. A competitive inhibition pattern was evident for vanadate ions on the activity of horseradish peroxidase (Ki = 41.2 microM). No significant inhibitory effects were found when V(V) was tested with catalase and when either V(IV) or V(V) were assayed with glutathione peroxidase. For the latter, the effect of V on the different components of the reaction system was investigated. V(V) did not significantly affect SOD activity when assayed with the sulfite method, which is devoid of interferences with V(V); however, there was an apparent inhibitory dose-response pattern for either V(IV) or V(V) using the pyrogallol assay, owing to an interference of pyrogallol with the metal. Besides, no significant binding of V(IV) or V(V) to the enzyme could be demonstrated. The lack of a direct inhibitory effect of V on the activity of the main antioxidant enzymes suggests that many biological and toxicological effects of V may be mediated more by oxidative reactions of the metal or of its complexes with physiologically relevant biomolecules than by a direct modulation of enzymatic activities.     

Biliary excretion and distribution of 51Cr(III) and 51Cr(VI) in rats

The biliary excretion and distribution of 51Cr after intravenous administration of 51Cr(III) (61CrCl5) or 51Cr(VI) (Na252CrO4 . 4 H2O) was studied in rats. The cumulative biliary excretion of 51Cr reached 24 hrs after the injection was significantly higher after administration of 51Cr(VI) than after 51Cr(III) 3.51+/-0.7% and 0.51+/-0.05% of administered dose, respectively). This difference was especially due to a higher rate of biliary excretion of 51Cr in the first hours after 51Cr(VI) administration. The excretion of 51Cr via faeces was also higher after administration of 51Cr(VI) (7.35+/-0.45%) OF ADMINISTERED DOSE, AS AGAINST 4.23+/-0.23% after 51Cr(III). On the other hand, no significant difference in urinary excretion of 51Cr was found. Statistically significant differences were also observed in the distribution of 51Cr in the organism after administration of both valence states of the metal.     

Biliary excretion of exogenous hematin in rats

Rats with bile fistula were injected intravenously with single doses of hematin (5,10,20,30, and 40 mg/kg body weight). Bile samples were collected every 30 min. for 4 hours, and at longer time intervals thereafter. The concentration of hematin in the bile was measured spectrophotometrically at 590 nm. The maximal hematin concentration in the bile (0.29 mg/ml for 40 mg/kg) observed 1.25 hours (average from 15 rats; range 0.5 – 1.5 hours) after hematin administration occured progressively later for lower doses of hematin, and for 5 mg/kg was observed at 3.14 hours (average for 4 rats; range 2–4 hours). The total hematin excreted within 4 hours was 8.0% of injected dose for 40 mg/kg and is dose dependent.     

The effect of some chelating agents on the biliary and urinary excretion of manganese in rats

The biliary excretion of manganese, in rats which have been loaded with manganese via their drinking water, can be significantly enhanced by the administration of the chelating agents: desferrioxamine (DSF), sodium bis(hydroxyethyl) dithiocarbamate (DEDTC), and sodium isonipecotamidedithiocarbamate (INADTC). The effect of these chelating agents on the urinary excretion of manganese was more complex and was found to be dependent upon the level of loading of manganese as well as the individual chelating agent. For animals with drinking water containing 400 mg/liter of manganese, the administration of the chelating agents led to a decrease in the sum of the biliary plus urinary manganese excretion. The results are of special interest in that they show that under some conditions the administration of chelating agents can lead to changes other than those expected.     

Hepatic uptake and biliary excretion of manganese in the little skate,Leucoraja erinacea

The liver is a major organ involved in regulating whole body manganese (Mn) homeostasis; however, the mechanisms of Mn transport across the hepatocyte basolateral and canalicular membranes remain poorly defined. To gain insight into these transport steps, the present study measured hepatic uptake and biliary excretion of Mn in an evolutionarily primitive marine vertebrate, the elasmobranch Leucoraja erinacea, the little skate. Mn was rapidly removed from the recirculating perfusate of isolated perfused skate livers in a dose-dependent fashion; however, only a small fraction was released into bile (< 2% in 6 h). Mn was also rapidly taken up by freshly isolated skate hepatocytes in culture. Mn uptake was inhibited by a variety of divalent metals, but not by cesium. Analysis of the concentration-dependence of Mn uptake revealed of two components, with apparent K_m values 1.1 ± 0.1 µM and 112 ± 29 µM. The K_m value for the high-affinity component was similar to the measured skate blood Mn concentration, 1.9 ± 0.5 µM. Mn uptake was reduced by nearly half when bicarbonate was removed from the culture medium, but was unaffected by a change in pH from 6.5 to 8.5, or by substitution of Na with Li or K. Mn efflux from the hepatocytes was also rapid, and was inhibited when cells were treated with 0.5 mM 2,4-dinitrophenol to deplete ATP levels. These data indicate that skate liver has efficient mechanisms for removing Mn from the sinusoidal circulation, whereas overall biliary excretion is low and appears to be mediated in part by an ATP-sensitive mechanism.     

The biliary excretion of trypsin in rats

The serum half-life of bovine [³H]acetyltrypsin was estimated to be 9 rain following intravenous administration in rats. This was maintained when six successive doses of 200 g each were given at 1-h intervals. The enzyme was removed from the circulation after complexing with 2-macroglobulin (2-M). The amount of³H label appearing in bile increased with each successive dose and this was associated with breakdown products (<10 000 daltons) of the ₂–M/[³H] acetyltrypsin. Intact –M/[³H] acetyltrypsin was recovered from bile but represented only 0.06% of the administered dose of active enzyme.     

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.     

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a 15N nuclear magnetic resonance study

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 A, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 A. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion.     

Activation of lignin and manganese peroxidase excretion in Phanerochaete chrysosporium by fungal extracts

Summary Lignin (LiP) and manganese peroxidase (MnP) excretion by Phanerochaete chrysosporium INA-12 was significantly increased in response to fungal extract supplementation. LiP and MnP production was increased 1.7- and 1.8-fold, respectively, with fungal extracts from agitated pellet cultures of strain INA-12, namely fungal extracts P₆ and P₄. In cultures supplemented with a fungal extract harvested from static cultures of strain INA-12 (fungal extract S₄), LiP and MnP production was increased 1.8- and 1.6-fold, respectively. Succinate dehydrogenase activity, a mitochondrial marker, was significantly enhanced (2.7-fold) in cultures with the addition of fungal extracts. Correspondence to: M. Asther     

Development of glutathione peroxidase activity during dietary and genetic copper deficiency

Copper deficiency was produced in developing rodents to study a possible interaction between copper and the selenoenzyme, glutathione peroxidase (GSH-Px). Dietary copper deficiency was investigated in Sprague-Dawley rats and in three mouse strains (C57BL, C3H/HeJ, C58); genetic copper deficiency was studied in two of the mouse strains, C57BL and C3H/HeJ, using brindled mice. In certain cases it appeared that copper deficiency was associated with depressed liver GSH-Px activity; values from copper-deficient livers were 40–70% of control values. However, the decrease in liver GSH-Px in both rats and mice was only observed when body weight was also depressed and did not necessarily correlate with copper deficiency signs, such as lower serum ceruloplasmin or liver cytochrome oxidase activities. In weanling rats, serum GSH-Px activity was normal despite a 60% reduction in liver activity. Mouse liver GSH-Px activity rose fourfold during the first 3 weeks of life to 75% of the adult level. Rat liver GSH-Px also increased during the suckling period. When perinatal copper deficiency, nutritional or genetic, was severe enough to retard growth, liver GSH-Px activity was depressed. Unlike rats, adult murine liver GSH-Px was equivalent in males and females.