Monolayer Cultures Of Rat Hepatocytes Research Articles

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

The level and compartmentalization of phosphatidate phosphatase-1 (lipin-1) control the assembly and secretion of hepatic VLDL

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.

Species-specific toxicity of diclofenac and troglitazone in primary human and rat hepatocytes

Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24 h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A – the major phase I enzymes involved in liver xenobiotic metabolism – we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.

Monolayer culture of primary rat hepatocytes on an Arg-Gly-Asp (RGD)-immobilized polystyrene dish express liver-specific functions of albumin production and p-acetamidophenol metabolism the same as for spheroid culture

The aim of this study was to evaluate the expression of albumin production and drug metabolism activities of primary rat hepatocytes under a two-dimensional monolayer culture condition formed on a RGD-immobilized culture dish. Albumin production activity on the RGD-immobilized dish (2500 ng cm −2 Pronectin F-coated dish) was 1.6 ( p < 0.05) and 1.2 ( p < 0.05) times higher than that on the collagen-coated dish and that of the spheroid culture, respectively. p-Acetamidophenol (APAP) metabolism rate of the collagen-monolayer at 5 days of culture was decreased to 16% of the activity at 1 day of culture. On the other hand, APAP metabolism activities of the RGD-monolayer and spheroid cultures were well maintained during 7 days of culture. However, we could not detect the effectiveness of the RGD-monolayer on lidocaine metabolism of hepatocytes. This is the first report suggesting that a hepatocyte monolayer on a RGD-immobilized culture substratum expresses albumin production and APAP metabolism activities equal to those of a hepatocyte spheroid culture.

Cytotoxicity of Fumonisin B1 in Spheroid and Monolayer Cultures of Rat Hepatocytes

Fumonisin B1 (FB1), the most prevalent member of toxins produced by several species of Fusarium molds, which occur mainly in maize, causes several fatal hepatopathies and nephropathies of animals. The current study was scrutinized to ascertain different cytotoxic and morphological transformations in rat hepatocytes induced by the treatments of diverse concentrations (300, 500, or 1000 μM) of fumonisin B1 in vitro, using both monolayer and spheroid cultures. In each hepatocyte culture, the cytotoxicity of FB1 was augmented in dose- and time-response manners. Morphological transformations among FB1-treated groups integrated accumulation of lipid droplets, cytoplasmic vacuolation in hepatocyte monolayers, and bleb formation in the hepatocyte spheroids. Additionally, electron microscopy revealed the loss of microvilli, mitochondrial swelling, and formation of lamellar membranous whorl in the vacuoles and bile canaliculi-like structures. Appearance of electron dense bodies in the monolayers, and loss of cell-to-cell contact in spheroids were depicted in 1000 μM FB1-treated hepatocytes. These outcomes insinuate different vital events in explaining morphological transformations in the cell membrane and organelles, induced by fumonisins in rat hepatocytes.

Hepatoprotective activity of Sapindus mukorossi and Rheum emodi extracts: In vitro and in vivo studies

To study the hepatoprotective capacity of Sapindus mukorossi (S. mukorossi) and Rheum emodi (R. emodi) extracts in CCl(4) treated male rats. The dried powder of S. mukorossi and R. emodi was extracted successively with petroleum ether, benzene, chloroform, and ethanol and concentrated in vacuum. Primary rat hepatocyte monolayer cultures were used for in vitro studies. In vivo, the hepatoprotective capacity of the extract of the fruit pericarp of S. mukorossi and the rhizomes of R. emodi was analyzed in liver injured CCl(4)-treated male rats. In vitro: primary hepatocytes monolayer cultures were treated with CCl(4) and extracts of S. mukorossi & R. emodi. A protective activity could be demonstrated in the CCl(4) damaged primary monolayer culture. In vivo: extracts of the fruit pericarp of S. mukorossi (2.5 mg/mL) and rhizomes of R. emodi (3.0 mg/mL) were found to have protective properties in rats with CCl(4) induced liver damage as judged from serum marker enzyme activities. The extracts of S. mukorossi and R. emodi do have a protective capacity both in vitro on primary hepatocytes cultures and in in vivo in a rat model of CCl(4) mediated liver injury.

A Metabolic Screening Study of Trichostatin A (TSA) and TSA-Like Histone Deacetylase Inhibitors in Rat and Human Primary Hepatocyte Cultures

Hydroxamic acid (HA)-based histone deacetylase (HDAC) inhibitors, with trichostatin A (TSA) as the reference compound, are potential antitumoral drugs and show promise in the creation of long-term primary cell cultures. However, their metabolic properties have barely been investigated. TSA is rapidly inactivated in rodents both in vitro and in vivo. We previously found that 5-(4-dimethylaminobenzoyl)aminovaleric acid hydroxyamide or 4-Me2N-BAVAH (compound 1) is metabolically more stable upon incubation with rat hepatocyte suspensions. In this study, we show that human hepatocytes also metabolize TSA more rapidly than compound 1 and that similar pathways are involved. Furthermore, structural analogs of compound 1 (compounds 2-9) are reported to have the same favorable metabolic properties. Removal of the dimethylamino substituent of compound 1 creates a very stable but 50% less potent inhibitor. Chain lengthening (4 to 5 carbon spacer) slightly improves both potency and metabolic stability, favoring HA reduction to hydrolysis. On the other hand, Calpha-unsaturation and spacer methylation not only reduce HDAC inhibition but also increase the rate of metabolic inactivation approximately 2-fold, mainly through HA reduction. However, in rat hepatocyte monolayer cultures, compound 1 is shown to be extensively metabolized by phase II conjugation. In conclusion, this study suggests that simple structural modifications of amide-linked TSA analogs can improve their phase I metabolic stability in both rat and human hepatocyte suspensions. Phase II glucuronidation, however, can compensate for their lower phase I metabolism in rat hepatocyte monolayers and could play a yet unidentified role in the determination of their in vivo clearance.

Azathioprine hepatotoxicity and the protective effect of liquorice and glycyrrhizic acid

This study aimed to evaluate the responses of human hepatocytes to azathioprine hepatotoxicity in comparison with the well-studied azathioprine hepatotoxicity in rat hepatocytes and the effects of protective agents to suppress azathioprine hepatotoxicity. Azathioprine presented its hepatotoxicity at clinically relevant concentrations (lower than 10 microm) in primary rat hepatocytes after 48 h of treatment as shown by a severe decrease in cell viability as well as intracellular GSH depletion. However, primary human hepatocytes exhibited only significant intracellular GSH depletion after treatment with azathioprine at these clinically relevant concentrations, while a reduction in cell viability by 29% was only evidenced after 48 h of treatment with azathioprine at the high concentration of 50 microm. In addition, a monolayer culture of primary rat hepatocytes was used as an in vitro model to examine the protective effects of antihepatotoxic drugs including glutathione (GSH), N-acetylcysteine (NAC, a GSH precursor), liquorice and glycyrrhizic acid (GA), a major bioactive component of liquorice, against hepatotoxicity of 1 microm azathioprine. It was found that both liquorice and GA showed substantial protection according to assays of cell viability and intracellular GSH, while neither GSH nor NAC had such a protective function. Similarly, GA protected human hepatocytes from intracellular GSH depletion on exposure to 1 microm azathioprine. These results implied that GA or liquorice could be considered as potent protection agents against azathioprine hepatotoxicity.

랫드 간세포 일차배양에서 양파 추출물이 수은에 의해 유도된 독성 및 지질과산화에 미치는 영향

본 연구의 목적은 랫드 간세포 일차배양에서 양파추출물이 수은에 의해 유발된 세포독성, 지질과산화 및 항산화효소 활성에 미치는 효과를 조사하기 위함이다. 수은 농도별 효과를 조사하기 위해 0, 1, 5, 10, 30 및 50 ppm의 다양한 농도의 <TEX>$HgCl_2$</TEX>로 6시간 동안 간세포를 일차배양하였다. 간세포 독성과 생존율은 배양액의 GOT와 LDH 활성 및 MTT 값으로 결정하였고, 지질과산화는 TBARS 농도로 측정하였다. 항산화에 미치는 효과는 catalase, GSH-Px, GSH-Rd 활성 및 DPPH free radical 소거활성으로 결정하였다. 10 ppm 이상의 <TEX>$HgCl_2$</TEX> 농도는 GOT 활성을 증가시켰고 %MTT 감소를 현저히 억제시켰으며 TBARS 농도를 증가시켰다. 또한 30 ppm 이상의 <TEX>$HgCl_2$</TEX> 농도는 LDH 활성을 증가시켜 수은이 농도 의존적으로 간세포 손상과 지질과산화를 촉진시켰다. 6시간 동안 30 ppm 농도의 <TEX>$HgCl_2$</TEX>, 처리는 간세포의 catalase, GSH-Px 및 GSH-Rd 활성을 현저히 감소시켰다. 수은 독성에 대한 양파추출물의 효과를 조사하기 위해 30 ppm의 수은 존재하에 0, 0.01, 0.05, 0.1 및 0.3 mg/ml의 다양한 농도의 양파추출물로 6시간 동안 간세포를 일차배양하였다. 0.05 mg/ml이상 농도의 양파추출물 첨가는 30 ppm 농도의 수은에 의해 증가된 GOT 활성을 감소시켰으며 MTT 감소를 증가시켰다. 또한 0.01 mg/ml 이상 농도의 양파추출물은 수은에 의해 증가된 LDH 활성과 TBARS 농도를 감소시켜 양파추출물이 수은에 의해 유발된 간손상과 지질과산화를 억제시켰음을 알 수 있었다. 0.05 mg/ml와 0.01 mg/ml 이상 농도의 양파추출물 첨가는 수은에 의해 각각 억제된 catalase와 GSH-Px 활성을 증가시켰으나 GSH-Rd 활성에는 영향을 미치지 못하였다. 양파추출물의 농도가 증가할수록 free radical 소거활성은 증가하여 5 mg/ml 농도의 양파추출물은 pyrogallol 용액의 흡광도 감소를 100%로 기준하였을 때 93% 이상의 소거 활성을 나타내었다. 이상과 같이 간세포 일차배양에서 수은은 간독성 증가, 간세포 생존율 감소, 지질과산화 촉진 및 catalase, GSH-Px, GSH-Rd 등의 항산화효소 활성 감소를 유발시켰으며, 양파추출물은 catalase와 GSH-Px의 활성을 증가시키고 free radical을 소거시켜 수은에 의해 유발된 독성 및 지질과산화를 억제함으로써 항산화 및 간보호 효과를 나타내는 것으로 사료된다. The objective of present study was to investigate the effect of onion extracts on mercuryinduced cytotoxicity, lipid peroxidation and antioxidant enzyme activities in primary monolayer cultures of rat hepatocytes. Primary cultures of rat hepatocytes were incubated for 6 hr in the presence of various concentrations (0, 1, 5, 10, 30 or 50 ppm) of <TEX>$HgCl_2$</TEX>. Cytotoxicity and cell viability were determined by measuring glutamic oxaloacetic transaminase (GOT) activity, lactate dehydrogenase (LDH) activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) value. Lipid peroxidation w as evaluated using thiobarbituric acid reactive substances (TBARS) assay. Effects of onion extract on antioxidant system were determined by measuring catalase, glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd) activities as well as DPPH free radical scavenging activity. <TEX>$HgCl_2$</TEX> at the concentration of 10 ppm increased GOT activity and TBARS concentration but decreased %MTT reduction, whereas <TEX>$HgCl_2$</TEX> at the concentration of 30 ppm increased LDH activity, representing that <TEX>$HgCl_2$</TEX> caused cytotoxicity and lipid peroxidation in dose-dependent manner, <TEX>$HgCl_2$</TEX> at the concentration of 30 ppm significantly decreased catalase, GSH-Px and GSH-Rd activities. When primary cultures of rat hepatocytes were incubated with various concentrations (0, 0.01, 0.05, 0.1 or 0.3 mg/ml) of onion extract for 6 hr in the presence of 30 ppm of <TEX>$HgCl_2$</TEX>, onion extracts at the concentration of 0.05 mg/ml decreased GOT activity, but increased %MTT reduction by 30 ppm of <TEX>$HgCl_2$</TEX>. <TEX>$HgCl_2-induced$</TEX> LDH activity and TBARS concentration were decreased by onion extract at the concentration of 0.01 mg/ml. Taken together, onion extract prevented H<TEX>$HgCl_2-induced$</TEX> hepatocyte injury and lipid peroxidation. Onion extracts at the concentration of 0.1 mg/ml almost or completely inhibited <TEX>$HgCl_2-induced$</TEX> catalase and GSB-Px activities. GSH-Rd activity, however, was not affected by onion extract. Free radical scavengjing activity was increased as concentration of onion extract increased. Onion extract at the concentrion of 5 mg/ml possesed mote than 93% scavenging activity comparing to 100% radical scavenging activity by pyrogallol solution as a reference. These results demonstrate that onion extracts suppressed mercury-induced cytoctoxicity and lipid peroxidation by scavenging free radical and increasing catalase and GSH-Px activities.

Heme-Reversible Impairment of CYP2B1/2 Induction in Heme-Depleted Rat Hepatocytes in Primary Culture: Translational Control by a Hepatic α-Subunit of the Eukaryotic Initiation Factor Kinase?

The role of heme in the phenobarbital-mediated induction of CYP2B1/2 was reexamined in rat hepatocytes in monolayer culture, acutely depleted of heme by treatment with either 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) or N-methylprotoporphyrins (NMPP). The findings revealed that such acute hepatic heme depletion markedly impaired CYP2B1/2 protein induction, an effect that was reversible by heme resupplementation. However, TaqMan analyses of hepatic mRNA isolated from these heme-depleted cells revealed that this impairment was not due to faulty transcriptional activation of either CYP2B1 or CYP2B2 gene expression as previously proposed, thereby confirming literature reports that heme is not a transcriptional regulator of the CYP2B1/2 gene. In contrast, the rate of de novo CYP2B1/2 protein synthesis was found to be dramatically inhibited in both DDEP- and NMPP-treated hepatocytes. Concurrently, a marked (>80%) suppression of de novo hepatocellular protein synthesis was also observed, along with a significantly enhanced phosphorylation of the alpha-subunit of the eukaryotic initiation factor eIF2 (eIF2alpha), as monitored by the phosphorylated eIF2alpha/total eIF2alpha ratio in these heme-depleted cells. Indeed, the parallel reversal of all these three effects by heme supplementation suggests that this impaired CYP2B1 induction most likely stems from blocked translational initiation resulting from the activation of a heme-sensitive eIF2alpha kinase. Such global suppression of hepatic protein synthesis may disrupt a myriad of vital cellular functions, thereby contributing to the clinical symptoms of acute hepatic heme-deficient states such as the hepatic porphyrias.

Prediction of in vitro intrinsic clearance from hepatocytes: comparison of suspensions and monolayer cultures.

Due to the time-dependent loss of cytochrome P450 (P450)-mediated metabolism in freshly isolated hepatocytes, several types of culture systems have been developed to extend their lifespan. The aim of this study was to evaluate the ability of monolayer cultures of rat hepatocytes to determine the in vitro CL(int) compared with suspensions of freshly isolated hepatocytes. Seven compounds were incubated in rat hepatocyte suspensions and monolayer cultures, and in vitro CL(int) was obtained via metabolite formation (12 pathways) or substrate depletion approaches. Only two compounds (tolbutamide and 7-ethoxycoumarin) gave comparable (within 2-fold) in vitro CL(int) in both suspensions and monolayer cultures. Although the overall rank order of compounds was the same in both models (covering a range of 3-4 orders of magnitude), the prediction of in vitro CL(int) for high-turnover compounds (seven pathways) was lower for monolayer cultures compared with suspensions, probably due to an uptake rate limitation leading to increases in K(M). In general, there was an average 50% loss of the P450 activity in monolayers based on a decrease in V(max) relative to suspensions. However, monolayer cultures gave a higher estimation of in vitro CL(int) for the low-turnover compound S-warfarin compared with fresh cell suspensions due to a decrease in the K(M) of the four individual metabolites. The use of hepatocyte monolayer cultures may offer the potential advantage of extending the lower end of the usable clearance range (below 0.1 microl/min/10(6) cells) for predicting in vivo CL(int).

CORRECTION TO "TISSUE DISTRIBUTION, STABILITY, AND PHARMACOKINETICS OF APO2 LIGAND/TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND IN HUMAN COLON CARCINOMA COLO205 TUMOR-BEARING MICE"

Due to the time-dependent loss of cytochrome P450 (P450)-mediated metabolism in freshly isolated hepatocytes, several types of culture systems have been developed to extend their lifespan. The aim of this study was to evaluate the ability of monolayer cultures of rat hepatocytes to determine the in vitro CL(int) compared with suspensions of freshly isolated hepatocytes. Seven compounds were incubated in rat hepatocyte suspensions and monolayer cultures, and in vitro CL(int) was obtained via metabolite formation (12 pathways) or substrate depletion approaches. Only two compounds (tolbutamide and 7-ethoxycoumarin) gave comparable (within 2-fold) in vitro CL(int) in both suspensions and monolayer cultures. Although the overall rank order of compounds was the same in both models (covering a range of 3-4 orders of magnitude), the prediction of in vitro CL(int) for high-turnover compounds (seven pathways) was lower for monolayer cultures compared with suspensions, probably due to an uptake rate limitation leading to increases in K(M). In general, there was an average 50% loss of the P450 activity in monolayers based on a decrease in V(max) relative to suspensions. However, monolayer cultures gave a higher estimation of in vitro CL(int) for the low-turnover compound S-warfarin compared with fresh cell suspensions due to a decrease in the K(M) of the four individual metabolites. The use of hepatocyte monolayer cultures may offer the potential advantage of extending the lower end of the usable clearance range (below 0.1 microl/min/10(6) cells) for predicting in vivo CL(int).

Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at −78 °C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME 2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME 2SO maintain 79.7 ± 6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7 ± 14.2% protein concentration, 55.4 ± 4.2% carboxyfluorescein diacetate de-acetylation, 27.2 ± 7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3 ± 7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.

Hepatocurative and antioxidant profile of HP-1, a polyherbal phytomedicine.

HP-1 a herbal formulation comprising of Phyllanthus niruri and extracts of Terminalia belerica, Terminalia chebula, Phyllanthus emblica and Tinospora cordifolia has been evaluated for hepatoprotective activity against carbon tetrachloride (CCl4) induced toxicity. Results show that HP-1 reversed the leakage of lactate dehydrogenase (LDH) and glutamate pyruvate transaminase (GPT) and prevented the depletion of glutathione (GSH) levels in a primary monolayer culture of rat hepatocytes (in vitro). HP-1 attenuated the serum toxicity as manifested in elevated levels of transaminases (glutamate oxaloacetate transaminase (GOT), and GPT) The antioxidative enzymes in liver (catalase and superoxide dismutase (SOD)) were restored to normal values after the oral administration of HP-1. HP-1 suppressed the formation of the superoxide anion radical and reduced CCl4 mediated lipid peroxidation (LPO). Silymarin and antioxidants (ascorbic acid, beta-carotene and alpha-tocopherol) were used for comparison. The present study showed that HP-1 is a potential hepatoprotective formulation with an additional attribute of being anti-peroxidative.

Damage to F-actin and cell death induced by chromium VI and nickel in primary monolayer cultures of rat hepatocytes

The toxicity of hexavalent chromium and nickel was investigated using primary cultures of hepatocytes as an in vitro system. Cr VI and Ni are widely used in the steel and orthopaedic implant industry. Although their toxicity has been extensively investigated, the mechanism(s) of action is/are not fully understood. Monolayer cultures of hepatocytes (10 5 cells/cm 2) were exposed to various concentrations of Cr VI and Ni for 24 h. Cells were stained with phalloidin-FITC for the detection of the cytoskeletal component, F-actin, and Annexin V-FITC and propidium iodide for the detection of the mode of cell death. Exposure of cells to Cr VI (1, 5, 10 and 50 μ m) resulted in the loss of the cell cytoskeleton, and this was accompanied by membrane blebbing and shrinking of the cell. Ni, on the other hand, induced detectable damage to the cytoskeleton only at 500 μ m. Staining of the cells with Annexin V and propidium iodide showed that Cr VI induces apoptosis at low concentrations (5 μ m), and necrosis at higher concentrations (25 and 50 μ m). Ni almost exclusively induced necrosis at 500 μ m with very few cells undergoing apoptosis. Below this concentration it had no discernable effect on hepatocytes. Damage to the cell cytoskeleton caused by Cr VI may be an early indication of apoptosis in hepatocytes.

Screening of various Swertia species extracts in primary monolayer cultures of rat hepatocytes against carbon tetrachloride- and paracetamol-induced toxicity

Swertia chirata Buch-Ham. (Gentianaceae), one of the oldest medicinal herbs of India, is a source of the Indian ayurvedic drug ‘chirata’ used for the treatment of liver disorders and malarial fevers. In this study, eight species of Swertia were collected. Each of the dry whole plant was extracted into methanol, the aqueous extract of which was sequentially extracted into hexane, chloroform and butanol extracts. The extracts were screened for their anti-hepatotoxic activity against carbon tetrachloride (CCl 4) and paracetamol (acetaminophen (AAP)) toxicity in primary monolayer cultures of rat hepatocytes. The primary cultures, 2.5×10 6 cells /3 ml medium/60 mm collagen-coated plates, were exposed to 2.5 mM CCl 4 or 12 mM AAP in the presence or absence of plant extracts (100 μg/ml culture medium). Cells and medium were harvested after 22 h of treatment for the assay of cellular reduced gluthathione (GSH) content and leakage of lactate dehydrogenase as biological end-points of toxicity. Both CCl 4 and AAP at the indicated concentrations reduced GSH by almost 50 and 80%, respectively, while the enzyme leakage was almost 15% above the untreated control. Hexane and methanol extracts of most of the species in general offered relatively good protection. The anti-hepatotoxic activity, nevertheless, was evident in all Swertia species against both the toxicants. However, Swertia purpurascens, Swertia chirata, Swertia paniculata and Swertia cordata exhibited better activity compared with other species investigated. In addition, influence of various extracts (10–100 μg/ml medium) was examined on cellular growth of rat Reuber hepatoma cell line H4IIEC3/G −. Except for the butanol extract of S. chirata, no other extracts exerted toxicity in terms of neutral red uptake by the cells.

Polyunsaturated Fatty Acids Suppress Hepatic Sterol Regulatory Element-binding Protein-1 Expression by Accelerating Transcript Decay

The reduction in hepatic abundance of sterol regulatory element binding protein-1 (SREBP-1) mRNA and protein associated with the ingestion of polyunsaturated fatty acids (PUFA) appears to be largely responsible for the PUFA-dependent inhibition of lipogenic gene transcription. Our initial studies indicated that the induction of SREBP-1 expression by insulin and glucose was blocked by PUFA. Nuclear run-on assays suggested PUFA reduced SREBP-1 mRNA by post-transcriptional mechanisms. In this report we demonstrate that PUFA enhance the decay of both SREBP-1a and -1c. When rat hepatocytes in monolayer culture were treated with albumin-bound 20:4(n-6) or 20:5(n-3) the half-life of total SREBP-1 mRNA was reduced by 50%. Ribonuclease protection assays revealed that the decay of SREBP-1c mRNA was more sensitive to PUFA than was SREBP-1a, i.e. the half-life of SREBP-1c and -1a was reduced from 10.0 to 4.6 h and 11.6 to 7.6 h, respectively. Interestingly, treating the hepatocytes with the translational inhibitor, cycloheximide, prevented the PUFA-dependent decay of SREBP-1. This suggests that SREBP-1 mRNA may need to undergo translation to enter the decay process, or that the decay process requires the synthesis of a rapidly turning over protein. Although the mechanism by which PUFA accelerate SREBP-1 mRNA decay remains to be determined, cloning and sequencing of the 3'-untranslated region for the rat SREBP-1 transcript revealed the presence of an A-U-rich region that is characteristic of a destablizing element.

The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures

It is important to assess the usefulness of long-term in vitro liver models for studying chronic toxicity, since acute assays may not reflect the in vivo situation. A potential long-term hepatocyte culture (i.e. liver spheroids) was investigated and compared to primary rat hepatocyte monolayer cultures following exposure to methotrexate (MTX), a well-documented chronic hepatotoxin. Following up to 7 days' treatment with MTX, cultures were morphologically assessed and assayed for enzyme leakage, intracellular reduced glutathione (GSH) and adenosine triphosphate (ATP). Spheroids maintained higher concentrations of GSH over the 14-day culture and ATP was maintained, but at a concentration not significantly different from monolayer cultures. Treatment of monolayer cultures resulted in concentration-related decreases in GSH and ATP, accompanied by enzyme leakage. In contrast, only ATP was affected following treatment of spheroids for 7 days. Spheroids appeared to be less sensitive to exposure to MTX, when compared with monolayer cultures. This may result from the maintenance of cellular functions, or from the lack of compound penetration into the three-dimensional spheroid structure. Therefore, the usefulness of spheroids to chronic in vitro toxicity testing may be limited.

Cultivation of rat hepatocytes in a medium based on commercially available infusate solutions

The possibility of using commercially available infusate solutions as a culture medium for hepatocytes was investigated in primary monolayer cultures of rat hepatocytes. The addition of Ca 2+ to the infusate medium was necessary for hepatocytes to express their albumin secreting ability. The infusate medium supplemented with hormones (10 −7 M insulin and 10 −7 M dexamethasone) and Ca 2+ (72.5 mg/ l) allowed hepatocytes to produce albumin of an amount comparable to that produced in Williams' E medium. The activity of released lactate dehydrogenase (LDH) was kept at a low level throughout the cultivation in the infusate medium.

Effect of 2,5-anhydro- d-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures

The fructose analogue 2,5-anhydro- d-mannitol (2,5-AM), which depletes liver cells of ATP, has been shown to alter liver cell membrane potential ( V m) in situ and in superfused liver slices. To study this effect of 2,5-AM on hepatocytes in more detail, patch-clamp experiments in the current-clamp mode were performed using two established models, rat hepatocyte couplets and confluent rat hepatocytes in primary culture. 2,5-AM, which has previously been shown to hyperpolarize hepatocytes in superfused liver slices and in vivo, failed to alter V m of hepatocyte couplets. Increasing intracellular Ca 2+ by addition of thapsigargin or ionomycin also did not evoke a change of V m. This is most likely due to a lack of Ca 2+-dependent K + channels in rat hepatocyte couplets. In contrast, 2,5-AM depolarized the cells in confluent hepatocyte monolayers. This depolarization was mimicked after inhibition of Na +/K + ATPase by ouabain. Ouabain was also able to block 2,5-AM’s effect on monolayer V m. Thus, 2,5-AM affects the membrane potential of isolated and cultured hepatocytes in a way not comparable with cells integrated in the liver.