Influence of copper-(II) on colloidal carbon-induced kupffer cell-dependent oxygen uptake in rat liver: Relation to hepatotoxicity

Formation of reactive O₂ species in biological systems can be accomplished by copper-(II) (Cu²⁺) catalysis, with the consequent cytotoxic response. We have evaluated the influence of Cu²⁺ on the respiratory activity of Kupffer cells in the perfused liver after colloidal carbon infusion. Studies were carried out in untreated rats and in animals pretreated with the Kupffer cell inactivator gadolinium chloride (GdCl₃) or with the metallothionein (MT) inducing agent zinc sulphate, and results were correlated with changes in liver sinusoidal efflux of lactate dehydrogenase (LDH) as an index of hepatotoxicity. In the concentration range of 0.1-1 μM, Cu²⁺ did not modify carbon phagocytosis by Kupffer cells, whereas the carbon-induced liver O₂ uptake showed a sigmoidal-type kinetics with a half-maximal concentration of 0.23 μM. Carbon-induced O₂ uptake occurred concomitantly with an increased LDH efflux, effects that were significantly correlated and abolished by GdCl₃ pretreatment or by MT induction. It is hypothesized that Cu²⁺ increases Kupffer cell-dependent O₂ utilization by promotion of the free radical processes related to the respiratory burst of activated liver macrophages, which may contribute to the concomitant development of hepatocellular injury.     

Expression of the novel protein PTPIP51 in rat liver: an immunohistochemical study

Expression of the novel protein tyrosine phosphatase interacting protein 51 (PTPIP51) was investigated on mRNA and protein level in the liver of adult Wistar rats. The presence of PTPIP51 mRNA was detected by Northern blotting. Immunostaining showed expression of PTPIP51 protein in distinct non-parenchymal cells. These cells were identified as Kupffer cells, stellate cells and natural killer cells by detection of cell-specific antigens. Whereas most endothelial cells lining large vessels reacted positive to the PTPIP51 antibody, sinusoidal endothelium showed no detectable amount of PTPIP51. Furthermore, PTPIP51 was also found to be expressed in cells forming the biliary tree. An additional subcellular analysis of the non-parenchymal cells by means of electron microscopy showed the presence of PTPIP51 protein in the cytoplasm and in the nuclei of non-parenchymal cells. Most of the hepatocytes did not show any immuno-detectable amount of PTPIP51, yet, some revealed PTPIP51 protein either in the cytoplasm or in the nucleus. This work is dedicated to Professor emeritus D. Sasse, Institute of Anatomy, University of Basel, Switzerland.     

Identification of G6PDH-active sinusoidal cells as Kupffer cells in the rat liver

Summary The aim of this study was to identify the G6PDH-active sinusoidal cells in the rat liver described by Rieder et al. (1978). Because of their number and distribution in the liver parenchyma, endothelial cells and pit cells could be excluded. Fat-storing cells were specifically marked by vital staining with vitamin A and identified by fluorescence microscopy. Kupffer cells could be detected after vital staining with carmine. Both staining methods allowed a subsequent incubation for the demonstration of G6PDH activity in the same unfixed cryostat section. Whereas more than 80% of the fluorescent particles were found outside the enzyme-positive cells, all G6PDH-active cells contained carmine particles. After counting the G6PDH-active cells, an estimation of 0.217 × 10⁸ cells/g liver tissue was obtained. The results indicate that high G6PDH activity is common to all Kupffer cells, and is therefore a highly specific marker enzyme for this class of sinusoidal liver cells.The essential parts of this study will be presented as an Inaugural-Dissertation to the Medical Faculty of the University of Freiburg by W. HosemannSupported by a grant from the SFB 46 (Molgrudent)     

Ethanol-dependent induction of bilirubin UDP-glucuronosyl-transferase in rat liver is mediated by Kupffer cells

Recently we have reported that bilirubin UDP-glucuronosyltransferase (UGT1A1) is induced in rat liver by chronic ethanol treatment. Several studies have shown that Kupffer cells play a central role in the mediation of various hepatic effects of chronic alcohol consumption. In the present work, the participation of Kupffer cells in the ethanol dependent induction of UGT1A1 was investigated. A group of rats was pretreated with gadolinium chloride, a known Kupffer-cell-depleting agent. We compared the effect of chronic ethanol ingestion on UGT1A1 expression in the liver of normal and gadolinium chloride treated rats. The effect of ethanol on bilirubin glucuronidation was completely prevented in Kupffer cell deficient rats. The western and northern blot analyses showed that the increase of both the protein and mRNA of UGT1A1 was prevented in these animals. These results suggest that Kupffer cells play a major role in the mediation of ethanol-stimulated induction of UGT1A1 in liver parenchymal cells.     

The action of n-propyl gallate on gluconeogenesis and oxygen uptake in the rat liver

In the present study the metabolic actions of n-propyl gallate on hepatic gluconeogenesis, oxygen uptake and related parameters were investigated. Experiments were done in the isolated perfused rat liver. n-Propyl gallate inhibited gluconeogenesis and stimulated oxygen uptake at concentrations up to 200 μM. The inhibitory effects on lactate gluconeogenesis (ED₅₀ 86.4 μM) and alanine gluconeogenesis were considerably more pronounced than those on glycerol and fructose gluconeogenesis. n-Propyl gallate also stimulated oxygen uptake in both the mitochondrial (63%) and microsomal (37%) electron transport chains. The first one is due mainly to the oxidation of n-propanol, as a metabolite of the first step of n-propyl gallate transformation. The second one results from a direct stimulation of the microsomal electron transport chain. n-Propyl gallate inhibited pyruvate carboxylation (ED₅₀ 142.2 μM) in consequence of an inhibition of pyruvate transport into the mitochondria an effect not found for gallic acid. This is probably the main cause for glucose output inhibition. Secondary causes are (1) deviation of intermediates for the production of NADPH to be used in microsomal electron transport; (2) deviation of glucose 6-phosphate for glucuronidation reactions; (3) gluconeogenesis inhibition by n-propanol, produced intracellularly from n-propyl gallate. Inhibition of mitochondrial energy metabolism is not significant in the range up to 200 μM, as indicated by the very small effect on the cellular ATP levels (5% decreased). n-Propyl gallate can be considered a kind of metabolic effector, whose actions on the liver metabolism are relatively mild although they can become harmful for the organ and the whole organism at high doses and concentrations.     

Studies on rat liver mitochondria: 4. Enzyme activities in mitochondria preserved at 0-4 degrees C

Rat liver mitochondria, stored with the energy-linked functions preserved or in aging conditions, were used to assay the activity of various enzymes during five days. The preservation of energy-linked functions was monitored by the respiratory control coefficient. ATPase, cytochrome oxidase and NADH dehydrogenase showed increased activity when the energy-linked functions were preserved. In aging conditions, cytochrome oxidase, NADH dehydrogenase and ATPase showed decreased activity. The ATPase activity increased only when mitochondria were stored in the presence of inhibitors of the electron transport chain. The activity of NADH oxidase did not change, and succinate oxidase and succinate dehydrogenase showed a small decrease in their activity. The enzymes of the matrix, alpha-ketoglutarate dehydrogenase, malate dehydrogenase and aspartate aminotransferase showed little decrease in activity under either of the conditions of storage. The total protein content decreased slightly under both conditions of storage. These results show that the activity of the enzymes analysed was maintained at reasonable levels, when the energy-linked functions of isolated mitochondria were preserved.     

Plasmodium yoelii: Influence of immune modulators on the development of the liver stage

Plasmodium sporozoites suppress the respiratory burst and antigen presentation of Kupffer cells, which are regarded as the portal of invasion into hepatocytes. It is not known whether immune modulation of Kupffer cells can affect the liver stage. In the present study, we found that sporozoites inoculated into Wistar rats could be detected in the liver, spleen, and lung; however, most sporozoites were arrested in the liver. Sporozoites were captured by Kupffer cells lined with endothelial cells in the liver sinusoid before hepatocyte invasion. Pretreatment with TLR3 agonist poly(I:C) and TLR2 agonist BCG primarily activated Kupffer cells, inhibiting the sporozoite development into the exoerythrocytic form, whereas Kupffer cell antagonists dexamethasone and cyclophosphamide promoted development of the liver stage. Our data suggests that sporozoite development into its exoerythrocytic form may be associated with Kupffer cell functional status. Immune modulation of Kupffer cells could be a promising strategy to prevent malaria parasite infection.     

Expression of P2X receptors on immune cells in the rat liver during postnatal development

Single and double-labeling immunofluorescence and RT-PCR expression of P2X receptor proteins and mRNAs were used in a study of the liver of postnatal rats. OX62 and ED1 were used as markers for dendritic and macrophage (Kupffer) cells respectively. The results showed that the P2X₆ receptor subunit was up-regulated by 15-fold on hepatic sinusoid cells during postnatal days P1 to P60. Subpopulations of Kupffer cells co-expressed P2X₄ and P2X₆ receptor subunits and dendritic cells co-expressed P2X₄ and P2X₇ receptor subunits. Lipopolysaccharide (endotoxin) injected into the peritoneal cavity led to increased expression of the P2X₆ receptor on Kupffer cells, suggesting that the P2X₆ receptor subunit may be up-regulated by endotoxin. This study presents the first evidence that P2X receptors are widely distributed in the rat liver immune system and that activation of Kupffer and dendritic cells in the rat liver might be regulated by extracellular ATP.     

Lack of recognition of Nepsilon-(carboxymethyl)lysine by the mouse liver reticulo-endothelial system: implications for pathophysiology

Advanced glycation end products (AGEs) are known to be associated with a number of pathological conditions, such as diabetes mellitus, Alzheimer's disease, uremia, as well as with normal aging. This study was undertaken to investigate whether Nepsilon-(carboxymethyl)lysine (CML), a major structure among numerous AGEs, engenders hepatic AGE clearance. For this purpose uptake of BSA substituted with heterogeneous AGEs or with CML only was monitored in vivo and in cultured hepatic scavenger cells. Here, we show that following intravenous administration of 125I-AGE-BSA and 125I-CML-BSA, blood radioactivity was reduced by 50% after 50s and >100 min, respectively. Recoveries from the circulation at 6 min after injection were: 5% for AGE-BSA, 95% for CML-BSA. More than 80% of the injected AGE-BSA was recovered from the liver. AGE-BSA, but not CML-BSA, was avidly endocytosed by cultured liver scavenger cells. Our results suggest that CML does not engender AGE-BSA clearance. Macromolecules substituted with CML only may escape elimination and cause pathological effects.     

Involvement of reactive oxygen species in the response of resistant (hypersensitive) or susceptible cowpeas to the cowpea rust fungus

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea ( Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus ( Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.     

Effect of mexiletine on glucose and oxygen uptake in rat brain, liver and myocardial tissue in vitro (author's transl)

The effect of mexiletine on oxygen and glucose consumption was studied both in homogenate and slices of brain, liver and myocardium of Wistar rats. Oxygen consumption was detected by means of Warburg's manometric techniques, and glucose utilization by the enzymatic method of glucose oxidase. Whilst glucose uptake was not modified in any of the studied preparations, mexiletine promoted a significant increase of oxygen consumption in the homogenized slices, and an inhibition in the intact tissue.     

溶解氧对虹鳟呼吸机能及红细胞特性的影响

目的：初步研究溶解氧对虹鳟呼吸机能及红细胞特性的影响。方法：设四组溶氧水平,测定呼吸频率、血液中PO2、PCO2和pH值,并以RBC、Hb、脆性、膜粘滞性及丙二醛含量等指标观察溶氧对红细胞的影响。结果：溶解氧升高使鱼呼吸频率降低,血液PO2升高（P〈0.05）;高溶氧导致红细胞数、丙二醛含量降低。红细胞膜粘滞系数在试验第10d时,高氧组明显高于其它组,但30d时各溶氧组差异不显著。结论：在一定范围内溶解氧升高可以改善机体组织氧含量,提高呼吸功能,红细胞对溶氧变化有其生理适应性。     

In vitro antitumour and hepatotoxicity profiles of Au(I) and Ag(I) bidentate pyridyl phosphine complexes and relationships to cellular uptake

In this study we characterised the in vitro antitumour and hepatotoxicity profiles of a series of Au(I) and Ag(I) bidentate phenyl and pyridyl complexes in a panel of cisplatin-resistant human ovarian cancer cell-lines, and in isolated rat hepatocytes. The gold and silver compounds overcame cisplatin-resistance in the CH1-cisR, 41M-cisR and SKOV-3 cell-lines, and showed cytotoxic potencies strongly correlated with their lipophilicity. Complexes with phenyl or 2-pyridyl ligands had high antitumour and hepatotoxic potency and low selectivity between different cell-lines. Their cytotoxicity profiles were similar to classic mitochondrial poisons and an example of this type of compound was shown to accumulate preferentially in the mitochondria of cancer cells in a manner that depended upon the mitochondrial membrane potential. In contrast, complexes with 3- or 4-pyridyl ligands had low antitumour and hepatotoxic potency and cytotoxicity profiles similar to 2-deoxy-D-glucose. In addition, they showed high selectivity between different cell-lines that was not attributable to variation in uptake in different cell-types. The in vitro hepatotoxic potency of the series of gold and silver compounds varied by over 61-fold and was closely related to their lipophilicity and hepatocyte uptake. In conclusion, Au(I) and Ag(I) bidendate pyridyl phosphine complexes demonstrate activity against cisplatin-resistant human cancer cells and in vitro cytotoxicity that strongly depends upon their lipophilicity.     

Multiplication stimulating activity (MSA) from the BRL 3A rat liver cell line: Relation to human somatomedins and insulin

The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component ot adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.     

Effects of retinoids on the production of tumour necrosis factor-alpha and nitric oxide by lipopolysaccharide-stimulated rat Kupffer cells in vitro: evidence for participation of retinoid X receptor signalling pathway

Kupffer cells play important roles in the development of liver injury by producing cytokines and free radicals. In consequence inhibition of these inflammatory mediators will be one of the targets for treating liver diseases. Retinoids modulate a wide variety of functions of monocytes/macrophages. Cellular effects of retinoids are mediated by two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We examined the effects of several kinds of natural and synthetic retinoids on the production of tumour necrosis factor-α (TNF-α) and nitric oxide (NO) by LPS-stimulated rat Kupffer cells in vitro. Of the various retinoids tested, 9-cis-retinoic acid (9-cis-RA) and Ro 13-6307, which are agonists of both RARs and RXRs, suppressed the production of TNF-α and NO in a concentration-dependent fashion, whereas three types of RAR-selective agonists, Ro 13-7410, Ro 40-6055 and Ro 19-0645 did not show any effect. Furthermore, the RARα antagonist, Ro 41-5253, did not prevent the effects induced by 9-cis-RA. The results suggest that these effects of 9-cis RA and Ro 13-6307 were induced by the RXRs-dependent signalling pathway. © 1997 John Wiley & Sons, Ltd.     

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

Protective effect of zinc against cadmium hepatotoxicity depends on this bioelement intake and level of cadmium exposure: a study in a rat model

It was estimated, in a rat model of moderate and relatively high chronic human exposure to cadmium (Cd), whether enhanced zinc (Zn) consumption may prevent Cd-induced liver injury and if the possible protective effect of this bioelement depends on its intake. For this purpose, the structure and function of the liver of the rats that received Zn (30 and 60 mg/l) or/and Cd (5 and 50 mg/l) for 6 months were evaluated. The treatment with Cd led to, dependent on the exposure level, pathological changes in the liver, including enhanced apoptosis and induction of inflammatory and necrotic processes. Moreover, the serum activities of hepatic marker enzymes (alanine transaminase and aspartate transaminase) and the concentration of proinflammatory cytokine – tumor necrosis factor α were increased. The supplementation with 30 and 60 mg Zn/l (enhancing daily Zn intake by 79% and 151%, respectively) partially or totally prevented from some of the Cd-induced changes in the liver structure and function; however, it provided no protection from necrosis, and the administration of 60 mg Zn/l during the higher Cd exposure even intensified this process. At both levels of Cd treatment, the use of 30 mg Zn/l was more effective in preventing liver injury than that of 60 mg Zn/l. The hepatoprotective impact of Zn may be explained, at least partly, by its antioxidative, antiapoptotic and anti-inflammatory action, ability to stimulate regenerative processes in the liver tissue, and indirect action resulting in a decrease in the liver pool of the non-metallothionein-bound Cd²⁺ ions able to exert toxic action. The results provide strong evidence that enhanced Zn consumption may be beneficial in protection from Cd hepatotoxicity; however, its excessive intake at relatively high exposure to Cd may intensify liver injury.     

Characteristics of Fe(II)ATP complex-induced damage to the rat liver mitochondrial membrane

It is well established that several iron complexes can induce oxidative damage in hepatic mitochondrial membranes by catalyzing the formation of ·OH radicals and/or by promoting lipid peroxidation. This is a relevant process for the molecular basis of iron overload diseases. The present work demonstrates that Fe(II)ATP complexes (5–50M) promote an oxygen consumption burst in a suspension of isolated rat liver mitochondria (either in the absence or presence of Antimycin A), caused mainly by lipid peroxidation. Fe(II)ATP alone induced small levels of oxygen uptake but no burst. The time course of Fe(II)ATP oxidation to Fe(III)ATP in the extramitochondrial media also reveals a simultaneous burst phase. The iron chelator Desferal (DFO) or the chain-break antioxidant butylated hydroxytoluene (BHT) fully prevented both lipid peroxidation (quantified as oxygen uptake burst) and mitochondrial swelling. DFO and BHT were capable of stopping the ongoing process of peroxidation at any point of their addition to the mitochondrial suspension. Conversely, DFO and BHT only halted the Fe(II)ATP-induced mitochondrial swelling at the onset of the process. Fe(II)ATP could also cause the collapse of mitochondrial potential, which was protected by BHT if added at the onset of the damaging process. These results, as well as correlation studies between peroxidation and mitochondrial swelling, suggest that a two phase process is occurring during Fe(II)ATP-induced mitochondrial damage: one dependent and another independent of lipid peroxidation. The involvement of lipid peroxidation in the overall process of mitochondrial membrane injury is discussed.Abbreviations AA Antimycin A - BHT butylated hydroxytoluene - EGTA ethylene glycol-bis(-aminoethyl ether) - N,N,N,N tetraacetic acid - DFO Desferal - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - SOD superoxide dismutase - TPP+ tetraphenylphosphonium bromide - TBARS thiobarbituric acid reactive substances