Toxicity assessments of chemical substances using primary culture of rat hepatocytes

A primary hepatocyte culture was used as a model system to assess the toxicity of various chemical substances. Chemical substances tested in this experiment included 14 kinds of organic solvents, arsenic acid and N-nitrosodiumethylamine, most of which are known as hepatotoxic materials. Enzyme (GPT and LDH) leakage and albumin secreted from the hepatocytes to culture medium were measured to evaluate the cell damage after exposure to the chemical substances at various concentrations for 3 d. Cell counts and protein contents before and after exposure to the chemical substances were also measured during the course of these experiments. The extent of LDH leakage from hepatocytes was parallel to that of GPT leakage after exposure to the chemicals. Albumin secreted from the hepatocytes in the culture medium evidently decreased at concentrations of the chemicals which increased the enzyme leakage. Viable cell counts were significantly decreased by chemicals that increased the enzyme leakage, although the cell protein contents were not significantly affected. The minimum concentration of the chemicals at which enzyme leakage from hepatocytes was significantly increased was defined as the lowest toxic concentration (TCL0). Judging from TCL0 values, the degree of toxicity in these chemicals seems to be almost identical to the degree of in vivo hepatotoxicity reported previously. We, furthermore, observed that there is a positive correlation (r = 0.780, p less than 0.01) between PT50 calculated by the LD50 value reported previously and -Log magnitude of TCL0.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of ethanol and calcium on fluid state of plasma membranes of rat hepatocytes

Basolateral (blLPM) and canalicular (cLPM) plasma membrane vesicles were isolated from rat liver to compare membrane fluidity, fluidity responses to membrane perturbants, and the relationship between fluidity and a membrane protein function such as carrier-mediated taurocholate transport. Membrane fluidity was measured by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene as a probe. Uptake of [3H] taurocholate was measured by a rapid Millipore filtration technique. blLPM were more fluid than cLPM. Ethanol produced a concentration-dependent fluidizing effect on both membrane preparations, the change being greater in blLPM. Incubation with calcium for 2 hr at 37 degrees C rendered both membrane preparations more rigid, again the cLPM being more resistant to perturbation. There was a linear correlation between an increase in membrane fluidity and inhibition of taurocholate uptake into blLPM in the presence of increasing concentrations of ethanol. The data support the concept that membrane lipid fluidity is an important regulator of membrane protein functions and hence also of overall cellular activity.     

Metabolic effects of ethanol on primary cell cultures of rat skeletal muscle.

Individuals who have consumed alcohol chronically accumulate glycogen in their skeletal muscles. Changes in the energy balance caused by alcohol consumption might lead to alcoholic myopathy. Experimental models used in the past, such as with skeletal muscle biopsy samples of alcohol-dependent individuals or in animal models, do not distinguish between direct effects and indirect effects (i.e., alterations to the nervous or endocrine system) of alcohol. In the current study, we evaluated the direct effect of ethanol on skeletal muscle glycogen concentrations and related glycolytic pathways. We measured the changes in metabolite concentrations and enzyme activities of carbohydrate metabolism in primary cell cultures of rat skeletal muscle exposed to ethanol for two periods. The concentrations of glycolytic metabolites and the activities of several enzymes that regulate glucose and glycogen metabolism were measured. After a short exposure to ethanol (6 h), glucose metabolism slowed. After 48 h of exposure, glycogen accumulation was observed.     

急性乙醇暴露对人原代肝细胞血红素氧化酶的影响

目的　研究急性乙醇暴露对人原代肝细胞血红素氧化酶活力及蛋白水平的影响。方法　经体外灌流、分离培养人原代肝细胞 ,观察乙醇对人原代肝细胞上清液中天冬氨酸氨基转移酶 (AST)的释放及谷胱甘肽 (GSH)含量的变化 ,用westernblot方法检测乙醇对人原代肝细胞血红素氧化酶活力及蛋白水平的影响。结果　急性乙醇暴露导致人原代肝细胞上清液中释放的AST增加 ,并呈明显的剂量效应和时间效应关系 ;此外 ,在 10 0mmol L乙醇 2 4h暴露下 ,肝细胞中的GSH明显降低 ,而HO 1酶活力在 0 5～ 12h之间明显升高 ,随后开始降低 ,且HO 1蛋白水平变化趋势与此一致。结论　HO 1酶活力的升高可能与乙醇暴露下人原代肝细胞氧化损伤的保护有关     

Diallyl trisulfide modulates cell viability and the antioxidation and detoxification systems of rat primary hepatocytes

This study investigated the effects of various concentrations of diallyl trisulfide (DATS) and incubation times on cell viability, glutathione (GSH) content, and GSH-related enzyme activity in rat primary hepatocytes. Isolated and cultured primary rat hepatocytes were used as an experimental model. Cells were treated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS for 0, 4, 8, or 24 h. After 24 h of treatment, some cells were incubated in fresh medium without DATS for an additional 24 h (48-h incubations). Based on lactate dehydrogenase (LDH) leakage and morphological examination, hepatocytes treated with 0.025 mmol/L DATS did not differ from the control cells at 4, 8, 24, and 48 h of incubation. However, LDH leakage was higher than in the control cells (P < 0.05) when the hepatocytes were treated with 0.05 or 0.25 mmol/L DATS for 4 h or more. The intracellular GSH levels of hepatocytes treated with 0.025 or 0.05 mmol/L DATS were higher than those of the control cells (P < 0.05), whereas those treated with 0.25 mmol/L DATS did not differ. The activity of glutathione reductase (GRd) was higher than in the control cells at 24 h (P < 0.05) when the hepatocytes were treated with 0.025 mmol/L DATS. When the hepatocytes were treated with 0.025 mmol/L DATS, the activity of glutathione S-transferase (GST) was higher than in the control cells at 48 h (P < 0.05). In hepatocytes treated with 0.05 mmol/L DATS, the activity of GST and glutathione peroxidase (GPx) was higher than in the control cells (P < 0.05) at 24 and 48 h of incubation. The results indicate that 0.025 or 0.05 mmol/L DATS could enhance antioxidation and detoxification capabilities by increasing the intracellular GSH level and the activity of GPx, GRd, or GST in rat primary hepatocytes. However, 0.05 or 0.25 mmol/L DATS might adversely affect the viability of hepatocytes.     

Cytotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin on primary cultures of adult rat hepatocytes

Primary cultures of adult rat hepatocytes were used to study the cytotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Rats were treated with a single oral dose of 5, 10 or 25Μg/kg of TCDD. At 2, 6, 10, 20, 30 days post-treatment, hepatocytes were isolated by a collagenase perfusion technique and maintained 48 hr as monolayers in serum-free medium in collagen-coated culture dishes. Uptake of ouabain was depressed in hepatocyte cultures isolated from all three doses of TCDD-treated rats. The effect was detected two days after TCDD-treatment and continued up to 30 days. The magnitude of the depression was directly related to the dose of TCDD. Hepatocyte cultures from TCDD-treated rats also showed depression in hormonally induced uptake ofα-aminoisobutyric acid (AIB) and tyrosine aminotransferase (TAT) activity at 10 and 25Μg/kg but not at 5Μg/kg. These cytotoxic effects of TCDD could be demonstrated only when TCDD was given to rats prior to isolation of hepatocytes; no toxic effects were observed when TCDD was added directly to the hepatocyte cultures. Pretreatment of rats with 1,3,6,8-tetrachlorodibenzo-p-dioxin had no effect on ouabain uptake, AIB transport, or TAT activities.     

Magnesium deficiency induces apoptosis in primary cultures of rat hepatocytes

The effects of extracellular magnesium (Mg) concentration on the rate of apoptosis in rat hepatocytes in primary culture were examined. After overnight attachment, incubations were conducted for up to 72 h in serum-free media containing low (0-0.4 mmol/L), physiological (0.8 mmol/L) or high (2 and 5.6 mmol/L) Mg concentrations. At 72 h, we observed numerous rounded hepatocytes on top of a shrunken cell monolayer at extracellular Mg concentrations < 0.8 mmol/L. These morphological features were associated with Mg-dependent differences in the total protein levels. The various Mg concentrations did not affect DNA synthesis; however, at a concentration < 0.8 mmol/L, the susceptibility of cultured rat hepatocytes to oxidative stress was increased as shown by the reduced glutathione concentration (10.6 +/- 2.8 vs. 37.3 +/- 4.1 nmol/mg protein with 0 and 0.8 mmol/L, respectively; P < 0.05) and increased lipid peroxidation (0.36 +/- 0.03 vs. 0.21 +/- 0.01 nmol malondialdehyde/mg protein with 0 and 0.8 mmol/L, respectively; P < 0.05). Fluorescence microscopy after Hoechst dye staining revealed numerous apoptotic figures in Mg-free monolayers compared with 0.8 and 5.6 mmol/L Mg conditions. These observations were confirmed quantitatively by flow-cytometric analysis after propidium iodide staining. The proportion of subdiploid cells decreased with increasing Mg concentration; for example, it was greater at 72 h in Mg-free cultures (76%) than in cultures containing 0.8 mmol/L or 5.6 mmol/L Mg (28%; P < 0.05). Caspase-3 was highly activated in Mg-free cultures after 48 h of treatment compared with 0.8 and 5.6 mmol/L conditions (P < 0.05). Overall, these results show that extracellular Mg deficiency has a negative effect on the survival of cultured rat hepatocytes by inducing apoptosis; however, supplementation of extracellular Mg did not reduce the spontaneous apoptosis that occurred over time in rat hepatocyte cultures.     

氟化物对原代培养大鼠肝细胞酶活力及超微结构的影响

目的研究不同剂量的氟化物对原代培养大鼠肝细胞存活率、酶活力及超微结构的影响。方法采用半原位胶原酶消化法分离大鼠肝细胞;MTT法检测细胞存活率;赖氏法检测培养液中谷草转氨酶(AST)和谷丙转氨酶(ALT)活性;透射电镜观察细胞超微结构改变。结果氟化钠染毒24小时后肝细胞存活率下降,且呈现明显的剂量-效应关系;2mmolL和4mmolL染氟组肝细胞培养液中ALT和AST的活性显著升高(P<005);透射电镜下染氟组肝细胞线粒体肿胀,内质网排列紊乱或断裂。结论过量氟化物对原代培养大鼠肝细胞有明显的毒作用,其主要作用方式是引起细胞膜和细胞器质膜损伤。     

Prenatal ethanol exposure alters the cytoskeleton and induces glycoprotein microheterogeneity in rat newborn hepatocytes

AIMS: Prenatal ethanol exposure (PEA) increases both liver weight and total protein content in the Golgi complex and alters its morphological and functional properties. As PEA-induced protein retention could be the synergetic consequence of alterations in the cytoskeleton and in the glycan biosynthesis, and there are no data that in liver PEA perturbs the cytoskeleton, we examined in hepatocytes whether PEA affects the main cytoskeleton elements. We also analysed whether ethanol induces glycoprotein microheterogeneity by altering the sugar composition of glycoproteins. METHODS: Livers from 0-day newborn control and PEA rats were used. The carbohydrate moiety of glycoproteins was determined by lectin blotting. The content and intracellular distribution of cytoskeleton proteins was analysed using immunoblotting, immunofluorescence and immunogold. RESULTS: PEA delayed the post-Golgi transport of albumin but not of transferrin. PEA also increased the levels of cytokeratin and tubulin, but it decreased the amount of tubulin capable of assembling into functional microtubules. PEA perturbed the distribution of cytokeratin and tubulin and induced microheterogeneity in several glycoproteins. CONCLUSIONS: PEA-induced retention of proteins in fetal hepatocytes could be the result of an alteration of glycoprotein biosynthesis and cytoskeleton-mediated transport.     

氟化钠对原代培养大鼠肝细胞的毒性作用

目的　探讨氟化钠 (NaF)对原代培养大鼠肝细胞的毒性作用。方法　采用原代培养的方法 ,观察氟化钠对原代培养大鼠肝细胞存活率和肝细胞培养液中谷草转氨酶 (AST)和谷丙转氨酶 (ALT)活性的影响。结果　氟化钠可使原代培养大鼠肝细胞存活率下降 ,且呈现明显的剂量 -效应关系 ,其IC50 为 3 5 8mmol/L ;肝细胞培养液中ALT和AST的活性随染毒剂量的增加而逐渐升高 ,2和 4mmol/L染氟组肝细胞培养液中ALT和AST的活性与对照组相比显著升高 (P <0 0 5 )。结论　氟化钠对原代培养大鼠肝细胞有明显的毒作用。     

Mechanism of radiation-induced diacylglycerol production in primary cultured rat hepatocytes.

Protein kinase C (PKC) is known to be a key enzyme in radiation-induced signal transduction pathways. We have previously demonstrated that gamma-irradiation induces PKC activation and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes (Int. J. Radiat. Biol. 70, 473-480, 1996). The present study was undertaken to investigate production of diacylglycerol, an endogenous activator of PKC, following gamma-irradiation of hepatocytes. Diacylglycerol content increased 3 min after irradiation, then decreased at 15 min and increased again at 30 min, indicating a biphasic pattern. This result implies participation of diacylglycerol in the radiation-induced activation of PKC in hepatocytes. In order to clarify the mechanism of the initial process of radiation-induced diacylglycerol production, the effects of reactive oxygens were investigated. Treatment of cells with hydroxyl radical, a major oxygen radical produced by radiation, induced diacylglycerol production without any change in the content of phosphatidylcholine, showing a peak at 1 min after treatment. No change in the diacylglycerol content was observed at that time by hydrogen peroxide treatment. Furthermore, the diacylglycerol production by hydroxyl radical was inhibited by pretreatment with neomycin sulfate, a phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor. These results suggest that radiation exerts PI-PLC activation through hydroxyl radical generation, followed by diacylglycerol production and PKC activation.     

Hormones and nutrients regulate acetyl-CoA carboxylase promoter I in rat primary hepatocytes

This study investigated the regulation of acetyl-CoA carboxylase (ACC) promoter activity by hormones and nutrients. Genomic clones including promoter I (PI) of the ACC gene were isolated and sequenced. ACC PI fragments (-1,049/+100 or -220/+21 bp) were subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. The ACC PI/luciferase chimeric plasmids were transfected into primary rat hepatocytes using lipofectin. Insulin treatment increased the activity of -1,049/+ 100 and -220/+21 ACC PI by 3.0- and 3.5-fold, respectively, compared to the control. The activity of both constructs was also increased by dexamethasone (Dex) and triiodothyronine (T3), with the greatest effects seen with all three hormones present. With -1,049/+100 or -220/+21 ACC PI, the addition of glucose increased luciferase activity compared to glucose-free control (p<0.05). On the other hand, polyunsaturated fatty acids (PUFA) reduced the activity of the -1,049/+100 ACC PI construct, with eicosapentaenoic acid and docosahexaenoic acid showing the greatest effect (about 70% of the control). However, the addition of PUFA to the culture media did not affect the activity of -220/+21 ACC PI. Therefore, insulin, Dex, T3, glucose, and PUFA regulate ACC gene expression, at least in part, through the PI promoter.     

N-acetylcysteine partially reverses oxidative stress and apoptosis exacerbated by Mg-deficiency culturing conditions in primary cultures of rat and human hepatocytes

OBJECTIVE: The effects of magnesium (Mg) deficiency on the rate of oxidative stress and apoptosis in primary cultures of human hepatocytes were compared to cultured rat hepatocytes. The possible reversion by N-acetylcysteine (NAC) in Mg-deficient culturing conditions was evaluated. METHODS: Incubations were conducted for up to 72 h in media containing a deficient (0-0.4 mM) or a physiological (0.8 mM) Mg concentration, and in the presence or absence of NAC after 24 h of culture in these Mg concentration conditions. RESULTS: We obtained similar profiles in terms of apoptosis and oxidative stress in primary cultures of human hepatocytes, as compared to rat hepatocytes, i.e. a Mg concentration-dependent effect on the caspase-3 activity and GSH levels after 72 h of culture, caspase-3 activity being highest and GSH levels being lowest in Mg-free cultures. The addition of NAC to culture media after the first 24 h of culture increased GSH concentrations. This was accompanied in Mg-deficient cultures by a decrease in both the caspase-3 activity and the lipid peroxidation. However, when culturing hepatocytes with physiological Mg concentrations, an increase in both caspase-3 activity and lipid peroxidation was observed. CONCLUSIONS: Our results indicate that Mg deficiency exacerbates the rate of apoptosis in cultured hepatocytes, associated with an increase in oxidative stress, the sensitivity of human hepatocytes being equivalent to that of rat hepatocytes. They also indicate a dual role of NAC and/or GSH, i.e. protective for hepatocytes placed in a Mg-deficient environment, while deleterious for hepatocytes placed in a Mg-physiological environment.     

Anti-inflammatory effects of daidzein on primary astroglial cell culture

Abstract

Introduction: Alzheimer's disease is the common cause of dementia in old people. The pathological hallmarks of Alzheimer's disease include neuronal loss, deposition of amyloid-β, and presence of neurofibrillary tangles. The endogenous steroid estrogen has been shown to affect neuronal growth, differentiation and survival, while isoflavones also have a neuroprotective effect on human cortical neurons. Daidzein, however, has a superior neuron-protective effect to other isoflavones. The present study is to determine whether daidzein is able to inhibit the production of pro-inflammatory mediators under amyloid-β and lipopolysaccharide stimulation.

Materials and methods: Astrocyte cells were stimulated with amyloid-β or lipopolysaccharide in the absence and presence of diadzein. Nitric oxide released into the culture media was determined using the Griess reaction, and concentrations of IL-1, IL-6, TNF-α and estrogen receptor gene expression were measured by semi-quantitative real-time polymerase chain reaction assay.

Results: Diadzein-treatment increases astrocyte cell counts and attains its maximal effect at the 10^?12M concentration. The addition of 20 μM amyloid-β or 10^?6 g/ml LPS can significantly decrease the viability of astrocytes, up-regulated IL-1, IL-6, TNF-α mRNA and estrogen receptor expression; in addition, 1-h daidzein pre-treatment can restore the decreased viability of astrocytes induced by amyloid-β or lipopolysaccharide as well as down-regulate their mRNA expression.

Conclusions: It seems that this response is estrogen receptor-mediated. These results further increase the possibility that daidzein may have potential to ameliorate the inflammatory process and also alleviate the risk of Alzheimer's disease progression.     

EROD activities in a primary cell culture of grass carp (Ctenopharyngodon idellus) hepatocytes exposed to polychlorinated aromatic hydrocarbonas

Aryl hydrocarbon (Ah) receptor (Ah-agonist) effects of environmental samples containing polychlorinated aromatic hydrocarbons were evaluated using a 7-ethoxyresorufin-O-deethylase (EROD) assay of a primary hepatocyte culture from grass carp (Ctenopharyngodon idellus). The results were compared with those obtained from the assay using the rat hepatoma cell line H4IIE and chemical analysis using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). A dose-response relationship was observed between the EROD activities, either from primary hepatocyte culture assay or from H4IIE assay, and concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that the assay based on the H4IIE cell line (EC50=0.83 pg/mL) is more sensitive to TCDD than the assay based on primary hepatocyte culture (EC50=9.7 pg/mL). In tests of environmental samples, the results from the assay using primary hepatocyte culture were comparable to those from the assay using the H4IIE cell line and chemical analysis of concentrations of mixtures of polychlorinated dibenzo-p-dioxin and dibenzofuran (PCDD/PCDF). The lack of a change in the activities of glutathione-S-transferase (GST) and lactate dehydrogenase (LDH) in cell culture upon exposure to TCDD indirectly indicates that the compound is persistent to biodegradation in the cell culture system.     

Acetylcarnitine-mediated inhibition of ethanol oxidation in hepatocytes

Carnitine-mediated prevention of ethanol-induced hepatic steatosis is related to the attenuation of ethanol metabolism by carnitine in the intact rat. Although carnitine retards ethanol oxidation in the intact animal, the in vitro activities of ethainol-metabolizing enzymes remain unaltered. Therefore, hepatocytes were targeted to understand the mechanism of carnitine effect on ethanol metabolism. Rat hepatocytes were isolated by a collagenase-perfusion technique and incubated in albumin-containing medium with ethanol in the presence or absence of added carnitine or related compounds. Ethanol oxidation was determined by the loss of ethanol as well as by the products formed. The rate of ethanol oxidation in the presence of carnitine was one-half the rate in the absence of carnitine (14 vs. 25 nmol · min⁻¹ · million⁻¹ cells). It took 100 times the concentration of carnitine to equal the maximal inhibition produced by acetylcarnitine and the effect of acetylcanitine was without a lag time. It is concluded that acetylcarnitine is the mediator of carnitine inhibition of ethanol oxidation.     

Toxic effect of cadmium stearate on rat cerebellum in culture

Summary Cadmium stearate inhibited the outgrowth of cerebellar cells from newborn rat in tissue culture and produced degenerative changes at a concentration of 0.58 × 10^–66 M, and the minimal dose for total inhibition of nerve fibers, glial cells, and fibroblasts was at about 2.3 × 10^–6 M.     

Alcohol-induced suppression of gluconeogenesis is greater in ethanol fed female rat hepatocytes than males.

The impact of alcohol-induced suppression on hepatic gluconeogenesis (HGN) after chronic ethanol consumption between males and females is unknown. To determine the effects of chronic alcohol consumption (8 weeks) on HGN, the isolated hepatocyte technique was used on 24 h fasted male and female Wistar rats. Livers were initially perfused with collagenase and the hepatocytes were isolated. Aliquots of the cell suspension were placed in Krebs-Henseleit buffer and incubated for 30 min with lactate, [U -14C]lactate, and nine different concentrations of ethanol (EtOH). Dose-effect curves were generated for the determination of maximal and half-maximal alcohol-induced inhibition on HGN. There was no significant difference in HGN (lactate only and no EtOH) between males and females fed the control diet (88.5 +/- 5.1 nmol/mg protein/30 min). Similarly, the HGN (lactate only and no EtOH) in males fed the ethanol diet (ME) were not significantly different (82.8 +/- 3.5 nmol/mg protein/30 min) compared to controls. In contrast, the females chronically fed the ethanol diet (FE) had significantly (P < .05) lower HGN (67.8 +/- 4.6 nmol/mg protein/30 min) compared to both ME and controls. With alcohol in the incubation medium, the HGN significantly (P<.05) declined in all groups. While alcohol suppressed HGN to a larger (P < .05) extent in ME (45.8 +/- 3.7 nmol/mg protein/30 min) compared to controls (64.0 +/- 3.8 nmol/mg protein/30 min), the inhibition was even greater (P < .05) in FE (32.7 +/- 3.2 nmol/mg protein/30 min). The more pronounced effect of chronic alcohol consumption on HGN in the presence of ethanol in female hepatocytes was supported by the concomitant decreases (P < .05) in 14C-lactate incorporation into 14C-glucose, lactate uptake, and 14C-lactate uptake. The results suggest that chronic alcohol consumption elicits a greater reduction on HGN in the presence of ethanol in the hepatocytes of females compared to males.     

Paradoxical effect of ethanol on liver lipogenesis in the genetically-obese Zucker rat

Sixteen obese (fa/fa) Zucker rats, sixteen lean (Fa/-) Zucker rats and sixteen Wistar rats, all male rats aged 7-8 weeks, were given either a control (C) diet containing no ethanol or an ethanol (E) diet in which 36% of the energy was supplied by ethanol, for a period of 4 weeks. The activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucose-6-phosphatase (EC 3.1.3.9) and glycerol kinase (EC 2.7.1.30) and the glycogen content in the livers of obese (fa/fa) rats were lower in animals given diet E than in those given diet C. As a result, hepatic lipogenesis and fatty degeneration of the liver were reduced in obese (fa/fa) rats given diet E.