Tanshinone IIA protects rat primary hepatocytes against carbon tetrachloride toxicity via inhibiting mitochondria permeability transition

Tanshinone IIA (Tan IIA), one of the key components of Salvia milthorrhiza Bunge (Lamiaceae), is used to treat liver disease. The present study was carried out to investigate the possible mechanisms involved in the hepatoprotective effects of Tan IIA on carbon tetrachloride (CCl₄)-induced hepatocyte toxicity. In cultures treated with 1 or 2 μM CCl₄, Tan IIA (10–75 μM) significantly increased hepatocyte survival rates. However, only at a concentration of 75 μM could Tan IIA partially reverse the CCl₄ (3 μM)-induced decrease of survival rate (34?±?3% vs. 18?±?3%, n?=?8, p?<?0.01). In isolated mitochondria energized with succinate, Tan IIA could inhibit the large swelling effect induced by CCl₄ (1 and 2 μM). Base on these results, Tan IIA could protect rat primary cultured hepatocytes from CCl₄-induced toxicity partially by the inhibitory effect on the opening of mitochondrial permeability transition (MPT).     

Dose-dependent toxicity of CCl4 in isolated rat hepatocytes and the effects of hepatoprotective treatments.

The temporal and dose-response relationships of the effects of CCl₄ were studied in isolated rat hepatocytes. CCl₄ decreased cell membrane integrity, as measured by the loss of alanine aminotransferase to the medium and the ability to retain intracellular K⁺. CCl₄ altered metabolic activity, as measured by the rate of urea synthesis and the lactate/pyruvate ratio. Addition of the following chemicals—SKF-525A, promethazine, dibenamine (0.1 mm); reduced glutathione (0.25 mm); hexobarbital methionine, cysteine (1 mm)—to the medium did not protect against the toxic effects of CCl₄, although these chemicals have been reported to be hepatoprotective in other experimental models. Incubation at 4°C did protect against CCl₄-induced changes in membrane integrity. These findings suggest that the isolated hepatocyte preparation is a suitable model for demonstrating time and dose-dependent toxic effects. However, they also suggest that there may be differences in mechanism compared with other experimental models.     

AMELIORIATION OF CHEMICALLY-INDUCED HEPATOCYTE INJURY BY CYCLOSPORINE A

Cyclosporine A, beside its current applications, possesses potential hepatoprotective effects. This study was directed to investigate the effect of Cyclosporine A pretreatment on hepatic injury due to carbon tetrachloride (CCl₄) and -galactosamine. Rats were injected by two successive doses of Cyclosporine A (5mgkg⁻¹day⁻¹). Six hours after the second dose, 1mlkg⁻¹of CCl₄was administered i.p. Effects associated with Cyclosporine A pretreatment were examined by using isolated hepatocytes and hepatocytes that were immobilized and continuously perfused. -Galactosamine (5m ) was added directly to the perfusion medium. After isolation, hepatocytes were examined histologically by light and electron microscopy, immobilized and perfused for further metabolic functional activity evaluation. Cyclosporine A pretreatmentin vivoproduced hepatoameliorative effects of various degrees which were statistically significant as manifested by: (1) an increased trypan blue exclusion after CCl₄; (2) an improved ureagenesis after CCl₄; (3) a reduction in the lipid droplets accumulation in the cytoplasm produced by CCl₄administration; (4) well preserved cytoplasmic organelles as mitochondria, endoplasmic reticulum ER, nuclear chromatin structures that were altered by CCl₄; and (5) an increased hepatocytes survival in the agarose gel matrix, reduction of LD leakage and improvement of ureagenesis after -galactosamine addition to the perfusion medium. The beneficial effect of Cyclosporine A pretreatment in modifying hepatotoxicity of chemical insults merits further studies.     

The effects of bromobenzene and Carbon tetrachloride exposure in vitro on the phospholipase C activity of rat liver cells

Homogenates were prepared from isolated rat hepatocytes, previously incubated (30 to 120 min) in the presence of bromobenzene (BB), Carbon tetrachloride (CCl₄), or dimethyl sulfoxide (DMSO = control vehicle). Homogenates of hepatocytes incubated with BB and CCl₄ compared to those exposed to DMSO exhibited a rapid increase (two- to five-fold) in their capacity to convert labeled, membrane-bound phosphatidic acid into diacylglycerol. The BB- and CCl₄-dependent increase in diacylglycerol (DG) content, at the expense of phosphatidate, was Ca²⁺ dependent and was attributed to changes in cell surface (plasma membrane) enzyme activity since the response occurred primarily in 1000g cell fractions. when either purified plasma membranes or 1000g cell fractions, isolated from fresh liver homogenates, were incubated with BB and CCl₄in vitro, a rise (two- to fourfold) in the conversion of labeled phosphatidate into DG was also observed. These Ca²⁺-dependent, BB- and CCl₄-induced changes in hepatocellular glycerolipid content were caused by a BB- and CCl₄-activated phospholipase C which degrades membrane phospholipids. This hypothesis is supported by the observation that ³²P-labeled phospholipids of hepatocyte monolayers were rapidly catabolized (10 to 50%) after BB and CCl₄ exposure (5 min). Also, there was an increased conversion of [methyl-¹⁴C]cytidine diphosphocholine into phosphatidylcholine after cell surface fractions were incubated with phospholipase C, BB, or CCl₄, a change which reflects a rise in hepatocellular DG content.A rapid, BB- and CCl₄-induced rise in hepatocellular phospholipase C activity that degrades cellular phospholipids could disrupt the structure and function of membranes and may represent an early event in toxic substance-related hepatocyte injury.     

Rubus sanctus protects against carbon tetrachloride‐induced toxicity in rat isolated hepatocytes: isolation and characterization of its galloylated flavonoids

Objectives Rubus sanctus Schreb., known from the Bible as ‘holy thorn bush’, grows wild in Egypt. Rubus sanctus aqueous alcoholic extract (RE) contains a complicated phenolic mixture (ellagitanins, flavonoids and caffeic acid derivatives). In this study, the phytochemical investigation of the plant was re‐evaluated. Herein, we report on the isolation and identification of three galloylated flavonoids, namely kaempferol‐3‐O‐(6″‐O‐galloyl)‐⁴C₁‐β‐d ‐galactopyranoside, quercetin‐3‐O‐(6″‐O‐galloyl)‐⁴C₁‐β‐d ‐galactopyranoside and myricetin‐3‐O‐(6″‐O‐galloyl)‐ C¹‐β‐d ‐galactopyranoside for the first time from the Rubus genus. We further aimed at evaluating the potential protective effects of RE against carbon tetrachloride (CCl₄)‐induced toxicity in isolated rat hepatocytes. Methods Based on an initial concentration‐response experiment, a concentration of 100 μg/ml was selected to investigate the hepatoprotective activity of RE. Key findings Pretreatment with RE afforded protection as indicated by counteracting CCl₄‐induced cell death, and reduced glutathione depletion. In addition, RE ameliorated CCl₄‐induced enzyme leakage by 40% for lactate dehydrogenase, 30% for alanine aminotransferase and 20% for aspartate aminotransferase as compared with CCl₄‐treated cells. Moreover, RE counteracted CCl₄‐induced lipid peroxidation and inhibited spontaneous lipid peroxidation in the control group. Conclusions In conclusion, RE protects against CCl₄‐induced toxicity in isolated rat hepatocytes.     

Anti-hepatotoxic activity of icariside II,a constituent ofEpimedium koreanum

Icariside II, a flavonol glycoside, was isolated from the aerial part ofEpimedium koreanum Nakai by the anti-hepatotoxic activity guided fractionation technique employing CCl₄-intoxicated primary cultured rat hepatocytes as an assay system. Its anti-hepatotoxic activity was evaluated by measuring activity of glutamic pyruvic transaminase released from the CCl₄-in-toxicated primary cultured rat hepatocytes. Icariside II significantly reduced the activity of glutamic pyruvic transaminase released from the CCl₄-intoxicated primary cultured rat hepatocytes and resulted in 78% recovery of the toxicity at the concentration of 200 μM. The antihepatotoxic activity of icariside II on the CCl₄-intoxicated primary cultured rat hepatocytes was as potent as that of silybin.     

Inhibition of TGFβ type I receptor activity facilitates liver regeneration upon acute CCl4 intoxication in mice

Liver exhibits a remarkable maintenance of functional homeostasis in the presence of a variety of damaging toxic factors. Tissue regeneration involves cell replenishment and extracellular matrix remodeling. Key regulator of homeostasis is the transforming growth factor-β (TGFβ) cytokine. To understand the role of TGFβ during liver regeneration, we used the single-dose carbon tetrachloride (CCl₄) treatment in mice as a model of acute liver damage. We combined this with in vivo inhibition of the TGFβ pathway by a small molecule inhibitor, LY364947, which targets the TGFβ type I receptor kinase [activin receptor-like kinase 5 (ALK5)] in hepatocytes but not in activated stellate cells. Co-administration of LY364947 inhibitor and CCl₄ toxic agent resulted in enhanced liver regeneration; cell proliferation (measured by PCNA, phosphorylated histone 3, p21) levels were increased in CCl₄ + LY364947 versus CCl₄-treated mice. Recovery of CCl₄-metabolizing enzyme CYP2E1 expression in hepatocytes is enhanced 7 days after CCl₄ intoxication in the mice that received also the TGFβ inhibitor. In summary, a small molecule inhibitor that blocks ALK5 downstream signaling and halts the cytostatic role of TGFβ pathway results in increased cell regeneration and improved liver function during acute liver damage. Thus, in vivo ALK5 modulation offers insight into the role of TGFβ, not only in matrix remodeling and fibrosis, but also in cell regeneration.     

Contributions of caspase-8 and -9 to liver injury from CYP2E1-produced metabolites of halogenated hydrocarbons

1.?Drug-induced liver injury is difficult to predict at the pre-clinical stage. This study aimed to clarify the roles of caspase-8 and -9 in CYP2E1 metabolite-induced liver injury in both rats and cell cultures in vitro treated with carbon tetrachloride (CCl₄), halothane or sevoflurane. The human hepatocarcinoma functional liver cell line was maintained in 3-dimensional culture alone or in co-culture with human acute monocytic leukemia cells.

2.?In vivo, laboratory indices of liver dysfunction and histology were normal after administration of sevoflurane. CCl₄ treatment increased blood AST/ALT levels, liver caspase-3 and -9 activities and liver malondialdehyde, accompanied by centrilobular hepatocyte necrosis. Halothane increased AST/ALT levels, caspase-3 and -8 activities (but not malondialdehyde) concomitant with widespread hepatotoxicity. In vitro, CCl₄ treatment increased caspase-9 activity and decreased both mitochondrial membrane potential (MMP) and cell viability. In co-culture, halothane increased caspase-8 activity and decreased MMP and cellular viability. There were no toxic responses in CYP2E1 knockdown in monoculture and co-culture.

3.?CYP2E1-inducing compounds play a pivotal role in halogenated hydrocarbon toxicity.

4.?Changes in hepatocyte caspase-8 and -9 activities could be novel biomarkers of metabolites causing DILI, and in pre-clinical development of new pharmaceuticals can predict nascent DILI in the clinical stage.     

Hepatoprotective activity of punarnavashtak kwath,an Ayurvedic formulation,against CCl4-induced hepatotoxicity in rats and on the HepG2 cell line

Objective: Punarnavashtak kwath (PNK) is a classical Ayurvedic formulation, mentioned in Ayurvedic literature Bhaishajya Ratnavali, for hepatic disorders and asthma. This study investigated the hepatoprotective activity of PNK to validate the traditional use of this formulation.

Materials and methods: PNK was prepared in the laboratory according to the method given in Ayurvedic literature. Phytochemical screening was performed to determine the presence of phytoconstituents. Hepatoprotective activity was evaluated against CCl₄-induced hepatotoxicity in rats and by its effect on the HepG2 cell line.

Results: Preliminary phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, saponins, and a bitter principle in PNK. Administration of PNK produced significant hepatoprotective effect as demonstrated by decreased levels of serum liver marker enzymes such as aspartate transaminase, serum alanine transaminase, serum alkaline phosphatase, and serum bilirubin and an increase in protein level. Thiopentone-induced sleeping time was also decreased in the PNK-treated animals compared with the CCl₄-treated group. It also showed antioxidant activity by increase in activity of glutathione, superoxide dismutase, and catalase and by a decrease in thiobarbituric acid reactive substance level compared with the CCl₄-treated group. Results of a histopathological study also support the hepatoprotective activity of PNK. Investigation carried out on the HepG2 cell line depicted significant increase in viability of cells exposed to PNK as compared with CCl₄-treated cells.

Discussion and Conclusion: It can be concluded that PNK protects hepatocytes from CCl₄-induced liver damages due to its antioxidant effect on hepatocytes. An in vitro study on HepG2 cell lines also supports its protective effect.     

Quantitative structure activity relationship for the acute cytotoxicity of 13 (bis)aziridinyl-benzoquinones: relation to cellular ATP depletion

This study was performed to establish relationships between the structure of 2,5-bis(1-aziridinyl)-1,4-benzoquinones (BABQs) bearing different substituents at the 3- and 6-position and their acute toxic effects in rat hepatocytes. The cell viability, loss of cellular glutathione (GSH_GSSG) and loss of ATP were followed during 4 h of incubation of freshly isolated hepatocytes. The toxicity of these compounds (100 μM) was predicted better by their reactivity with GSH than by their redox cycling in rat liver microsomes. The time of 50% loss of viability (LT₅₀) correlated very well with the time of 50% depletion of ATP (AT₅₀). LT₅₀ could be adequately predicted by using the electronic field parameter (F_total) describing the electron withdrawing or donating properties for all the substituents on the quinone-nucleus. 7-(Di)halogen-substituted BABQs that all very rapidly depleted cellular glutathione showed significant differences in AT₅₀ as well as in LT₅₀. This suggests that alterations in ATP levels are important for explaining the differences in cytotoxicity of these compounds.     

Production of volatile hydrocarbons by isolated hepatocytes: An in vitro model for lipid peroxidation studies

Increased production of pentane, ethane, ethylene, and thiobarbituric acidreactive substances were measured in hepatocytes treated with bromotrichloromethane (CCl₃Br), carbon tetrachloride, and chloroform. Linear correlations between these indices of lipid peroxidation were found when CCl₃Br-treated hepatocytes still had high viability. Of the halogenated hydrocarbons tested, CCl₃Br was the most potent initiator of peroxidation and caused the greatest amount of cellular damage as measured by trypan blue dye exclusion and glutamate pyruvate transaminase released from the cells. Hepatocytes from livers of rats fed a semisynthetic vitamin E-supplemented diet were slightly more protected from peroxidation and cellular damage induced by CCl₃Br treatment than were hepatocytes from rats fed a vitamin E-deficient diet. The usefulness of isolated hepatocytes as a model for lipid peroxidation was demonstrated.     

Metabolism of acrylamide to glycidamide and their cytotoxicity in isolated rat hepatocytes: protective effects of GSH precursors

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in rodents. The recent discovery of AA at ppm levels in a wide variety of commonly consumed foods has energized research efforts worldwide to define toxicity and prevention. Metabolism and cytotoxicity of AA and its epoxide glycidamide (GA) were studied in the hepatocytes freshly isolated from male Sprague–Dawley rats. The isolated hepatocytes metabolized AA to GA. The formation of GA followed Michaelis-Menten kinetic parameters yielded apparent K _m = 0.477 ± 0.100 and 0.263 ± 0.016 mM, V _max = 6.5 ± 2.1 and 26.4 ± 3.0 nmol/h/10⁶ cells, and CL_int = 14 ± 5 and 100 ± 12 μl/h/10⁶ cells for the hepatocytes from untreated and acetone-treated rats, respectively. There were lower K _m and marked increases in V _max (fourfold) and in CL_int (sevenfold) in acetone-treated rat hepatocytes. The data suggest that CYP2E1 played a major role in metabolizing AA to more toxic GA. Both AA and GA induced a concentration- and time-dependent glutathione (GSH) depletion of the hepatocytes. From decreasing rates of GSH contents in hepatocytes, the parameters of glutathione S-transferase (GST) in hepatocytes to AA and GA were calculated to be K _m = 1.4 and 1.5 mM, V _max = 21 and 33 nmol/h/10⁶ cells, and CL_int = 15 and 23 μl/h/10⁶ cells, respectively. GA 1.5-times more readily depleted GSH content than AA. GA decreased the viability of hepatocytes at 3 mM, but AA did not. These data indicate that GA is more toxic than AA as assessed by intracellular GSH depletion and loss of viability of hepatocytes. GSH precursors such as N-acetylcysteine and methionine provided significant anti-cytotoxic effects on the decrease of GSH content and cell viability of hepatocytes induced by GA and AA.     

Hepatoprotective activity of Feronia limonia root

Objectives The aim of this study was to evaluate the hepatoprotective potential of a methanolic extract and of marmesin isolated from the root bark of Feronia limonia. Methods Activity levels of aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT), cell viability and cell death were evaluated in HepG2 cells (human liver hepatoma cells) treated with CCl₄ in the presence or absence of F. limonia extract or marmesin. Plasma activity levels of AST, ALT, bilirubin, alkaline phosphatase, protein, hepatic antioxidants, lipid peroxidation and histopathological evaluations were carried out in rats treated with CCl₄ alone or co‐supplemented with F. limonia extract or marmesin in a dose‐dependent manner. Key findings In‐vitro co‐supplementation of F. limonia methanolic extract or marmesin significantly minimized alteration in levels of AST and ALT and improved cell viability. Oral administration of F. limonia methanolic extract or marmesin significantly prevented CCl₄‐induced elevation in the plasma markers of hepatic damage and hepatic lipid peroxidation and a decrease in hepatic antioxidants. In‐vivo hepatoprotective potential of F. limonia methanolic extract and marmesin was evident from the minimal alterations in the histoarchitecture of liver. Conclusions This has been the first scientific report on the hepatoprotective potential of F. limonia root bark methanolic extract and marmesin.     

Effect of Di-(2-ethylhexyl) Phthalate on Rat Liver Injured by Chronic Carbon Tetrachloride Treatment

Abstract The effect of Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, was studied using histopathological and biochemical parameters on rat liver injured by carbon tetrachloride (CCl₄). The mild centrilobular necrosis observed with CCl₄ (7.7 mmol/kg subcutaneously and biweekly up to 38 days) and mild congestion and bile duct proliferation produced by DEHP (2.5 mmol/kg intraperitoneally daily for ten days after the day 28 of experiment) were modified into extensive necrosis of the parenchymal cells when the animals received both chemicals. Groups of hepatocytes mostly at the periphery of the lobules also showed coagulative necrosis and some central and portal veins were completely occluded. Alterations in the activity of serum and liver enzymes of the animals receiving both chemicals were not significantly different from those treated with CCL alone, except in case of glucose-6-phosphatase (G-6-Pase) and SGPT. The characteristic decrease of G-6-Pase and increase of SGPT was less marked. Although the exact mechanism of the chemical interaction between CCl₄ and DEHP is not known, the results indicate their combined toxic potentiality.     

Genoprotective and hepatoprotective effects of Guarana (Paullinia cupana Mart. var. sorbilis) on CCl4-induced liver damage in rats

Context: Several biological effects of Paullinia cupana (guarana) have been demonstrated, but little information is available on its effects on the liver. Objective: The current study was designed to evaluate the hepatoprotective and genoprotective effects of powder seeds from guarana on CCl₄-induced liver injury in rats. Materials and methods: Male Wistar rats were pretreated with guarana powder (100, 300 and 600?mg/kg) or silymarin 100?mg/kg daily for 14 days before treatment with a single dose of CCl₄ (50% CCl₄, 1?mL/kg, intraperitoneally). Results: The treatment with CCl₄ significantly increased the serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). In addition, CCl₄ increased the DNA damage index in hepatocytes. Guarana in all concentrations was effective in decreasing the ALT and AST activities when compared with the CCl₄-treated group. The treatment with guarana decreased DNA damage index when compared with the CCl₄-treated group. In addition, the DNA damage index showed a significant positive correlation with AST and ALT. Discussion and conclusion: These results indicate that the guarana has hepatoprotective activity and prevents the DNA strand breakage in the CCl₄-induced liver damage in rats.     

CCl4-induced lipid peroxidation in isolated rat hepatocytes with different oxygen concentrations

It has been difficult to demonstrate convincingly the occurrence of lipid peroxidation by increases in the concentration of thiobarbituric acid (TBA) reactants in response to carbon tetrachloride (CCl₄) in hepatocytes isolated from fed, nontreated rats. This study was undertaken to determine whether or not the incubation atmosphere and the content of the incubation media could be contributing factors for the inhibition of CCl₄-induced lipid peroxidation. Isolated hepatocytes incubated in either Eagle's minimum essential medium (EMEM) or Tris buffer were incubated with and without CCl₄ (1 or 5 μl/4 ml hepatocyte suspension) under an atmosphere of either air or carbogen (95% O₂:5% CO₂). Samples were taken at 30, 60, or 120 min for determination of ethane (in the gas phase), TBA reactants concentration, and leakage of lactate dehydrogenase (LDH). Under air, CCl₄ stimulated ethane formation and increased the concentration of TBA reactants in hepatocytes isolated from fed, nontreated rats. This stimulatory effect was found irrespective of the incubation medium, although absolute values were generally lower in Tris buffer than in EMEM. Losses of LDH were also observed with the higher concentration of CCl₄. When carbogen was the atmosphere for incubation, no significant increases in the lipid peroxidation parameters could be found in response to CCl₄. Again, this response was irrespective of the incubation medium. Under carbogen, CCl₄-induced LDH leakage was no longer observed. Thus, these data provide a possible explanation as to why it has been difficult to observe lipid peroxidation, as evidenced by elevated TBA reactants concentration in hepatocytes isolated from fed, nontreated rats.     

Functional Impairment of Sinusoidal Membrane Transport of Organic Cations in Rats with CCl4-Induced Hepatic Failure

Purpose. The effect of CCl₄-induced experimental hepatic failure(EHF) on the sequential hepatobiliary transport of model organiccations (OCs), triethylmethylammonium (TEMA), and tributylmethylammonium (TBuMA), was investigated in rats. Methods. EHF was induced by an i.p. injection of CCl₄ at a dose of1 ml/kg 24 hr prior to the transport study. The cumulative in vivobiliary excretion, in vitro hepatic uptake by isolated hepatocytes,in vitro efflux (i.e., release) from hepatocytes, andin vivo hepatobiliary excretion clearance were measured for normal and CCl₄-EHF rats. Results. The CCl₄-EHF decreased the apparentin vivo biliary clearance (CL_o) and the in vitromaximum uptake rate (V_{max, uptake}) of TBuMA by 66 and 48%,respectively. The CCl₄-EHF had no effect on the CL_oof TEMA, but decreased both the V_{max, uptake} (59%) and thein vitromaximum hepatic efflux rate (Vmax, efflux) of TEMA (80%). On thecontrary, the CCl₄-EHF had no influence on the in vivohepatobiliaryexcretion clearance (CL_exc) of both OCs. Conclusions. Transport systems for the OCs on the sinusoidal membrane(uptake and/or efflux), rather than those on the bile canalicularmembrane (excretion) appear to be prone to damage by the CCl₄-EHF.     

Mechanism of Early Carbon Tetrachloride Toxicity in Cultured Rat Hepatocytes

Abstract: CCl₄ causes liver necrosis in a dose-dependent manner in vivo. However, we found that primary rat hepatocytes in culture were killed after a 2 hr incubation with carbon tetrachloride gas at CCl₄ partial pressures above a threshold between 45 and 54 mmHg. Below this threshold concentration no increase in hepatocyte death was observed. We sought to explain the very abrupt CCl₄ concentration threshold for hepatocyte death. Two inhibitors of cytochrome P450 2E1, cimetidine and diallyl sulfide, inhibited lipid peroxidation as measured by production of isoprostanes, but did not reduce hepatocyte death from CCl₄. At 37°, CCl₄ accelerated the mitochondrial permeability transition in vitro, at a threshold CCl₄ concentration similar to that which caused hepatocyte death. Phospholipase A₂ inhibitors, mepacrine and 4-bromophenacyl bromide, inhibited the increase in mitochondrial permeability, but did not inhibit hepatocyte death caused by CCl₄. Rat liver microsomal lipids were used to make liposomes loaded with Ponceau Red (FW 760.6). No leakage of Ponceau red was found at CCl₄ concentrations greater than the threshold for cell death. However, CCl₄ caused acceleration of liposome fusion, over the CCl₄ concentration range spanning the threshold for hepatocyte death. Early hepatocyte death in cell culture is independent of metabolism of CCl₄, and may be related to direct effects of CCl₄ on intracellular membranes.     

Toxicity studies in isolated hepatocytes from selenium-deficient rats and vitamin E-deficient rats

Isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats were treated with cumene hydroperoxide (CuOOH), phorone (diisopropylene acetone), acetaminophen, and diquat. The effect of these chemicals on cell viability, glutathione synthesis and release, and lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances was determined during a 4-hr incubation in a complete medium under 95% O₂:5% CO₂ at 37°C. CuOOH-treated (0.5 mm) selenium-deficient and vitamin E-deficient hepatocytes lost viability sooner than control hepatocytes. Thus, loss of selenium or vitamin E from the hepatocyte resulted in a cell more susceptible to damage by CuOOH. Phorone treatment (1.65 mm) resulted in depletion of intracellular glutathione in all three groups to approximately 20% of that in untreated hepatocytes. Cell viability and TBA-reactive substances were the same in treated and untreated hepatocytes. Thus, lowering of intracellular glutathione did not result in the spontaneous loss of cell viability or increased lipid peroxidation in selenium-deficient or in vitamin E-deficient hepatocytes. Acetaminophen appeared to be less toxic to selenium-deficient hepatocytes than to controls. This finding is in agreement with whole animal studies reported previously showing that selenium deficiency protects rats against acetaminophen hepatotoxicity. A potential explanation of this result is stimulation of glutathione synthesis by selenium deficiency. Severely vitamin E-deficient hepatocytes were protected from cell death by 12.5 and 25.0 mm acetaminophe, apparently by its antioxidant properties, while 50.0 mm acetaminophen was toxic to them. At all concentrations used, acetaminophen decreased the TBA-reactive substances present in the hepatocyte suspensions. Diquat (0.1 mm) caused more rapid cell death and higher levels of TBA-reactive substances in selenium-deficient hepatocytes than in control hepatocytes. Diquat toxicity in selenium-deficient isolated hepatocytes was not as severe as its toxicity in selenium-deficient whole animals, however.     

Hepatoprotection of 1β-hydroxyeuscaphic acid – the major constituent from Rubus aleaefolius against CCl4-induced injury in hepatocytes cells

Abstract

Context: Rubus aleaefolius Poir. (Rosaceae) is used as a folk medicine to treat various types of hepatitis with significant effects in Fujian Province of China. The ethyl acetate fraction of R. aleaefolius root ethanol extract proved effective after our testing in vivo animal experiments.

Objective: The protective effects of a major constituent, 1β-hydroxyeuscaphic acid isolated from R. aleaefolius was first investigated against carbon tetrachloride (CCl₄)-induced injury in liver cells from hepatocytes cell line (BRL-3A).

Materials and methods: Treatment of BRL-3A with CCl₄ led to generation of free radicals detected after a 2?h incubation and produced cell injury demonstrated by increased leakage of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) in the media. Exposure to CCl₄ caused apoptosis to cells but did not induce lipid peroxidation. Following treatment with 1β-hydroxyeuscaphic acid at doses ranging from 1 to 100?µg/mL for 24?h, cellular morphology, cell growth function (MTT assay), ALT, AST, malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed and evaluated under control and exposed conditions.

Results: The IC₅₀ of 1β-hydroxyeuscaphic acid was 15?μg/mL. Exposure of injured BRL-3A at 20?μg/mL changed abnormal size, cellular shrinkage, and enhanced regulation. ALT, AST, MDA enzyme levels were reduced and SOD activity was increased.

Discussion and conclusion: Treatment with 1β-hydroxyeuscaphic acid has significant hepatoprotective activity by lowering the leakage of intracellular enzymes, reducing the oxidation of proteins and decreasing the incidence of apoptosis. These results provide a basis for confirming the traditional uses of R. aleaefolius in treating hepatic diseases.