Cytochemical localization of glutathione in isolated and cultured rat hepatocytes

Summary A method for rapid staining of glutathione in cultured cells is described. This staining procedure is compatible with plastic culture dishes.     

3H-corticosterone reception by rat hepatocytes

Laboratory of Molecular Mechanisms of Intercellular Interactions, Institute of Biochemistry, Siberian Branch, Russian Academy of Medical Sciences, Novosibirsk. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 10, pp. 362–364, October, 1992.     

Developmental changes of proteoglycan synthesis in rat liver and isolated hepatocytes

The synthesis of sulfated proteoglycans in late fetal (19th to 22nd day of intrauterine life), early postnatal, and adult liver tissue as well as in hepatocytes and their distribution in plasma membranes were studied. Overall proteoglycan production is enhanced two-fold in fetal as compared with adult liver tissue. In contrast to slices from adult liver, in which the synthesis of heparan [35S]-sulfate comprises more than 80% and chondroitin sulfate less than 5% of total glycosaminoglycans, chondroitin [35S]sulfate is the major type of glycosaminoglycans synthesized in fetal liver representing about 50% of total sulfated glycosaminoglycans. Thus, the synthesis of chondroitin sulfate is elevated nearly 30-fold in fetal liver as compared with the adult counterpart. Immediately after birth chondroitin sulfate formation decreases rapidly reaching adult levels between the 10th and 15th day of postnatal life. The production of heparan sulfate is almost unchanged during perinatal liver development due to a relatively low fractional synthesis of heparan [35S]sulfate in fetal liver. Hepatocytes were identified as the cell type responsible for elevated chondroitin sulfate production in fetal liver. Erythroblasts, which synthesize chondroitin sulfate, contribute less than 10% to total glycosaminoglycan synthesis in embryonic liver. Plasma membranes of adult liver contain exclusively heparan sulfate whereas in neonatal liver cell membranes 25% of labeled glycosaminoglycans is represented by chondroitin sulfate, a fraction which decreases rapidly after birth. In parallel to the postnatal shut down of chondroitin sulfate synthesis the activity of the UDPxylose:coreprotein xylosyltransferase (EC. 2.4.2.26) decreases from 4.8 +/- 0.5 dpm/h per microgram protein to 0.3 +/- 0.1 dpm/h per microgram protein suggesting a regulatory function of the enzyme for proteochondroitin sulfate synthesis in developing liver. The formation of both heparan sulfate and chondroitin sulfate is dependent on functioning protein synthesis, which indicates, together with double labeling experiments using [3H]serine and [14C]glucosamine as isotopic precursors, their synthesis as proteoglycans. The positive correlation (r = 0.949) between the incorporation of [3H]thymidine into DNA and chondroitin [35S]sulfate production supports the assumption of a cell growth promoting activity of chondroitin sulfate and points to a significant role of the glycosaminoglycan in the process of cellular proliferation and tissue differentiation.     

组胺对大鼠正常肝细胞及缺氧/复氧时肝细胞活力的影响

目的:评价组胺对大鼠正常肝细胞及缺氧/复氧时肝细胞活力的影响。方法:大鼠肝细胞株,调整细胞密度为1×107cells/L,接种于培养孔中,每孔200μL作为研究对象,本研究包括2个实验,各实验中研究对象随机分为7组。实验1:空白组和2×104μg/L组各12孔,其它各组各6孔,空白组不做任何处理,10μg/L组、102μg/L组、103μg/L组、104μg/L组、2×104μg/L组和5×104μg/L组分别加入相应浓度的组胺,孵育25h,各组取6孔,测定肝细胞活力,取空白组和2×104μg/L组的其它6孔,收集细胞培养上清液和细胞,分别用于测定谷丙转氨酶(ALT)和超氧化物歧化酶(SOD)的活性,电镜观察细胞超微结构。实验2:分组方法同实验1,空白组和2×104μg/L组各12孔,其它各组各6孔,空白组不做任何处理,加入相应浓度的组胺后立即进行缺氧1h、复氧24h,各组取6孔,测定肝细胞活力,取空白组和2×104μg/L的其它6孔,收集细胞培养上清液和细胞,分别用于测定ALT和SOD的活性,电镜观察细胞超微结构。结果:实验1:与空白组比较,103μg/L组-5×104μg/L组肝细胞活力增加,且呈浓度依赖性,2×104μg/L组SOD活性升高(P0.05),ALT活性差异无显著(P0.05);电镜下空白组和2×104μg/L组肝细胞未见损伤。实验2:与空白组比较,102μg/L组-5×104μg/L组肝细胞活力降低,其中2×104μg/L组和5×104μg/L组肝细胞活力降低更明显,2×104μg/L组ALT活性升高,SOD活性降低(P0.05),2×104μg/L肝细胞损伤重于空白组。结论:组胺可增加大鼠正常肝细胞活力,且呈浓度依赖性,2×104μg/L组胺可提高其抗氧化能力;组胺可降低缺氧/复氧时大鼠肝细胞活力,且与浓度有关,2×104μg/L组胺可降低其抗氧化能力。     

d-Amphetamine-induced cytotoxicity and oxidative stress in isolated rat hepatocytes

Amphetamines (AMP) are potent psychostimulants and commonly used drugs of abuse. Its chronic administration creates tolerance and addiction and also associated with neurotoxicity and hepatocellular damage through oxidative stress. The present study was designed to evaluate the cytotoxic effects as well as the oxidative stress induced by d-amphetamines in isolated rat hepatocytes. Hepatocytes were isolated by collagenase perfusion technique and were exposed to different concentrations of AMP (0.2, 0.4, 0.8 and 1.6 mM) in a time-course experiment for up to 2 h. AMP exposure induced a significant decrease in cell viability and a significant increase in the leakage of hepatic enzymes {lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and asparate aminotransferase (AST)} in a concentration and time-related manner. In the same experiment, GSH content and thiobarbituric acid reactive substances (TBARS) generation were determined as indices of oxidative stress and lipid peroxidation respectively. AMP exposure results in a significant decrease in cellular GSH content as well as a significant enhancement of TBARS accumulation in a concentration and time-related manners. The obtained results suggested that 2-h exposure of hepatocytes to AMP (0.8 mM) was accompanied by submaximal responses. Therefore, a subsequent dose–response experiment was designed to evaluate the role of GSH modulation and oxidative stress in AMP toxicity in hepatocytes at 2 h. LDH release and TBARS generation were used as indicators in this experiment. Pretreatment with the GSH-depleting agents, chlorodinitrobenzene (CDNB), buthionine sulfoximine (BSO), or bis(chloroethyl)-nitrosurea (BCNU) enhanced the cytotoxicity of AMP. Conversely, pretreatment with GSH or sulfhydryl compounds such as methionine (MT), cysteine (CYS) or dithiothreitol (DTT) attenuated AMP toxicity. Similarly, co-incubation with enzymatic antioxidants, superoxide dismutase (SOD) or catalase (CAT) or iron chelator, desferroxiamine (DFO) or the hydroxyl radical scavengers, dimethylsulfoxide (DMSO) exhibited significant protection against AMP cytotoxicity. The present results indicate that AMP has a potential cytotoxic effect in isolated rat hepatocytes. AMP cytotoxicity is concentration-dependent. GSH depletion and oxidative stress play an important role in enhancing hepatotoxic potential of AMP in isolated rat hepatocyte. Thiol group-donors, antioxidants, free radical scavengers and iron chelators can play a critical role against AMP-induced cellular damage.     

Parameters of Adenylate Pool as Predictors of Energy Metabolism Disturbances in Hepatocytes during Hypoxia

We studied the dependence of various parameters of adenylate pool in hepatocytes on oxygen concentrations. Isolated cells responded to a decrease in oxygen content in their microenvironment by changes in components of the adenine nucleotide system, which attested to phasic nature of this process. Three ranges of oxygen concentrations differing by the type of changes in the parameters of adenylate pool were distinguished: steady-state range of these parameters; primary changes in the adenylate pool aimed at minimization of energy losses (compensatory stage characteristic of the initial stages of hypoxia); and linear drop of ATP content paralleled by decompensation of the regulatory mechanisms of ATP formation and adenine nucleotide degradation. Hence, parameters of the adenylate pool can serve as predictors of different stages of hypoxia. Differences in the parameters of adenylate pool depending on the level of O₂ in hepatocytes of rats highly and low-resistant to hypoxia indicate that energy metabolism is a mechanism involved in the formation of individual cell resistance to oxygen deficiency. These data suggest that suspension of isolated hepatocytes as an adequate cellular model for experimental studies of the effects of hypoxia on energy metabolism and functional activity of the cell.     

Agglutination kinetics of normal and diabetic adult rat hepatocytes

This paper describes Concanavalin A-induced agglutionation of viable rat hepatocytes obtained by collagenase perfusion from normal and streptozocin-treated diabetic rats. An irreversible cell-to-cell agglutination model is proposed to explain hepatocyte flocculation. The rate of agglutination is concentration dependent with respect to Concanavalin A, and is twice as fast in normal as compared to diabetic hepatocytes. Sticking probability constants ranging from 18.48×10⁷ to 4.6×10⁷ cm^–1 · hepatocyte^–1 and 8.32×10⁷ to 2.31×10⁷ cm^–1 · hepatocyte^–1 are calculated as a measure of agglutination for normal and diabetic cells respectively. These findings suggest that the cytoplasmic membranes of normal cells possess agglutinin receptors which are more numerous and/or differently arranged than those existing in diabetic cells.     

Postnatal development of myocardial cells after oxygen deficiency in utero

Pregnant rats were kept at a simulated altitude hypoxia (5000 m = pO2 11.38 kPa) for eight hours every day from the 16th to 21st day of pregnancy. The heart musculature of the left ventricle of newborn male rats underwent electron microscopic qualitative and morphometric quantitative examinations on the 2nd, 5th, 11th and 22nd day. Prenatal hypoxia leads to delayed development of the heart. The reduced weight on the 2nd day compared with that of control animals is compensated on the 22nd day. Prenatal hypoxia causes matrix disintegration and cristolysis of the mitochondria. Volume density, which is lower on the 2nd day (0.24: 0.29 (control animals], reaches that of the controls by the 22nd day. But the specific surface density of the cristae mitochondriales is markedly higher in the animals subjected to hypoxia (2nd day 31.0: 20.6; 22nd day 44.0: 38.1). Prenatal hypoxia causes an increase, in the postnatal period, in the autophagocytosis processes, which normally already exist in the heart musculature; this is recognizable by an increase in the volume density of the autophagic vacuoles (2nd day 0.0049: 0.0034; 22nd day 0.0098: 0.0045). The changes observed are regarded as reversible, i.e. as adaptive processes.     

Improved sensitivity of the unscheduled DNA synthesis assay in primary rat hepatocytes following culture in serum-free defined media

The unscheduled DNA synthesis (UDS) assay has been used extensively for the in vitro detection of DNA damage caused by compound exposure. However, the in vitro UDS assay has been insensitive for the detection of certain chemicals, particularly nitroaromatic compounds, that are positive in bacterial mutation assays. Recently, studies have been reported which describe alterations in the he-patocyte membrane following collagenase perfusion. Independently, a method for serum-free tissue culture has been developed which results in the up-regulation of cell surface receptors and which may restore membrane functions. Fourteen compounds, including seven nitroaromatics, were evaluated in the in vitro UDS assay employing a serum-free procedure. Five compounds that were previously reported positive in the standard in vitro UDS assay were also found positive using the serum-free method. In addition, five of the nitroaromatic compounds produced positive results with the serum-free method. 1-Methyl-3-nitro-1-nitrosoguanidine and 2-acetylaminofluorene, routinely used as positive controls in the UDS assay, showed greater activity in the serum-free assay. These results suggest that the use of serum-free media improves the sensitivity of the in vitro UDS assay and that the serum-free procedure potentially offers an effective alternative to the more labor intensive and more costly in vivo UDS assay. © 1995 Wiley-Liss, Inc.     

Intracellular changes in rat hepatocytes after intratracheal administration of highly dispersed silicon dioxide and uridine effects on these changes

Rat hepatocytes were examined under electron microscope at early terms after intratracheal administration of highly dispersed silicon dioxide powder against the background of uridine treatment. Penetration of powder particles into hepatocyte cytoplasm, nuclei, mitochondria, and peroxisomes and development of bacteria in these cells were observed. Uridine reduced the destructive effect of powder on the organelles, increased glycogen content in hepatocytes, and inhibited the formation of capsulated bacterial forms in these cells. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 596–600, May, 2006     

Effect of complexes of apolipoprotein A-I with tetrahydrocortisol and pregnenolone on protein biosynthesis in rat hepatocytes culture

Complexes of apolipoprotein A-I with tetrahydrocortisol and pregnenolone exhibit high biological activity and increase the rate of protein biosynthesis in the culture of rat hepatocytes. An important role in this process is played by reduced Δ⁴-3-keto group in the A-ring of steroid hormones. A complex of apolipoprotein A-I and pregnenolone modulated the rate of protein biosynthesis in liver cells. Hence, the observed changes are not organ-specific for this steroid. Our results suggest that this mechanism of regulation play an important role in intracellular regeneration and proliferation. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 9, pp. 264–266, September, 2007     

Effects of antioxidants on survival of adult rat hepatocytes under various oxygen tensions in serum-free primary culture.

Effects of antioxidants, such as superoxide dismutase, vitamin C, vitamin E, 4-(0-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane), and selenite on survival of adult rat hepatocytes were examined under normoxic and hyperoxic conditions in serum-free primary culture. The tested antioxidants, except for vitamin C, significantly increased the survival rate of hepatocytes under the normoxic condition (under air). Thus, even the normoxic culture condition is hyperoxic for hepatocytes. Elevation of oxygen tension (40% O2) caused severe morphologic degeneration of hepatocytes and remarkable decrease in the survival rate of the cells. Addition of the antioxidants effectively protected hepatocytes from the morphologic degeneration, and significantly improved the survival of the cells under the hyperoxic condition. These findings indicate that the antioxidants can maintain the long-term survival of hepatocytes in serum-free primary culture.     

Semi-automatic counting of connexin 32s immunolocalized in cultured fetal rat hepatocytes using image processing

Connexin 32s (Cx32s) were immunolocalized in fetal rat hepatocytes and their distribution was determined qualitatively. We used image analysis using a quantitative index (QI) of Cx32 (QI Cx32) defined as the area of Cx32s/number of cells in cultured fetal rat hepatocytes. Hepatocytes from livers of fetal rats were separated by collagenase digestion and low centrifugation on gestational day 17. Cells were cultured for 3 days in dexamethasone (DEX)-supplemented medium (Dex0). The medium was replaced with fresh medium and cells were continuously cultured for 3 days with DEX or epidermal growth factor supplemented medium (Dex3 or EGF3). After culture termination, cells were fixed and stained using the fluorescein-labeled antibody method for Cx32s and diaminophenylindole staining for nuclei. Thirty pairs of histological images for Cx32s and nuclei, 180 images in total, were obtained from each condition. The QI Cx32 significantly increased from 284.1 ± 102.0 (mean and SD, n = 26) of Dex0 to 428.9 ± 101.0 of Dex3 (n = 28) (P < 0.05, Kruskal-Wallis test, then Steel-Dwass test). The increase of QI Cx32 was compatible with the morphological observations. The image analysis processing time after preparation for 180 images was reduced from 8 h needed for manual operations to 1 min using ImageJ software with our macro routine.