Stress In Rat Hepatocytes Research Articles

Biochemical Evaluation of Adansonia Digitata Methanolic Leaf and Stem Bark Extract on Streptozotocin Induced Diabetic Rats

Several herbal preparations are used to treat diabetes; however, their overall biochemical effects other than hypoglycemic effects are scanty. This study therefore was designed to evaluate the effect of methanolic extract of leaves and stem-bark of Adansonia digitata on biochemical indices of streptozotocin-induced diabetic rats. Sixty-three wistar rats were distributed into 9 groups of 7 each. The test animals were intraperitoneally administered with single dose of 50 mg/kg streptozotocin and monitored for 72 hours for development of hyperglycemia. Diabetic rats were treated in 12 h cycles for three weeks with 100 mg/kg, 200 mg/kg and 300 mg/kg of both leaves and stem-bark of Adansonia digitata methanolic extract and metformin (50 mg/kg). Non-diabetic control rats received a lacebo of distilled water. Group 1 served as normal control, group 2 served as diabetic control, while groups 4-9 were diabetic rats treated orally with methanolic leave and stem-bark extracts of Adansonia digitata (100 mg/kg, 200 mg/kg and 300 mg/kg) for 21days. Group 3 animals were diabetic rats treated with anti-diabetic drug (metformin 50 mg/kg). The levels of fasting blood sugar (FBS), electrolytes (Na+, K+, Cl-), Urea and creatinine Triglyceride (TG), Cholesterol (CHOL), thiobarbituric acid reactive substance (TBARS), White Blood Cell (WBC) count, Red Blood Cell (RBC) count, Haemoglobin (HGB), Hematocrit (HCT), Platelet (PLT), Mean Cell Volume (MCV), Mean Cell Haemoglobin (MCH), Mean Cell Haemoglobin Concentration (MCHC), Lymphocyte (LYM), aspartatate aminotransfrease (AST), alanine aminotransferase (ALT), total protein (TP), albumin (ALB), catalase (CAT) and superoxide dismutase (SOD) activities total, total bilirubin (TB), direct bilirubin(DB) and indirect bilirubin (IB) concentrations were assayed. The results indicated that the concentrations of Na+, K+, Cl-, creatinine, urea TBARS, ALT, AST, ALB, CHOL and IB bilirubin were significantly (p<0.05) increased, while the levels of WBC, RBC, HGB, HCT, PLT, LYM, MCV SOD and CAT were reduced in the diabetic control (p<0.05). The Adansonia digitata methanolic leaves and stem-bark extract significantly increased WBC, RBC, HGB, HCT, PLT, MCV, LYM, TP, CAT and SOD activity and reduced the FBS, Na+, K+, Cl-, TBARS, ALT, AST, creatinine, urea and bilirubin concentrations significantly (p<0.05) compared to normal control. However, treatment with metformin showed slight modification in the changes observed compared to Adansonia digitata methanolic leaves and stem-bark extract. MCV and MCHC reduced non-significantly (p>0.05) in the diabetic animals as compared to the normal control and the extract-treated rats, while they increased non- significantly (p>0.05) in the test groups when compared to the diabetic control. Levels of TB, DB, and TG showed non-significant (p<0.05) increase in diabetic control, but treatment with extracts and metformin caused non-significant decrease (p<0.05). Diabetic control exhibited significantly (p<0.05) decreased ALB levels and non-significant TP decrease compared to normal control, while Adansonia digitata extracts and metformin significantly increased ALB and non-significantly increased TP levels compared to diabetic control. The study concluded that Adansonia digitata extracts reversed diabetes-induced oxidative stress in rat hepatocytes, potentially through beta cell regeneration or insulin release stimulation, suggesting their potential for managing diabetic complications.

Preventive effects of fructose and N-acetyl-L-cysteine against cytotoxicity induced by the psychoactive compounds N-methyl-5-(2-aminopropyl)benzofuran and 3,4-methylenedioxy-N-methamphetamine in isolated rat hepatocytes.

Psychoactive compounds, N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB) and 3,4-methylenedioxy-N-methamphetamine (MDMA), are known to be hepatotoxic in humans and/or experimental animals. As previous studies suggested that these compounds elicited cytotoxicity via mitochondrial dysfunction and/or oxidative stress in rat hepatocytes, the protective effects of fructose and N-acetyl-l-cysteine (NAC) on 5-MAPB- and MDMA-induced toxicity were studied in rat hepatocytes. These drugs caused not only concentration-dependent (0-4mm) and time-dependent (0-3hours) cell death accompanied by the depletion of cellular levels of adenosine triphosphate (ATP) and glutathione (reduced form; GSH) but also an increase in the oxidized form of GSH. The toxic effects of 5-MAPB were greater than those of MDMA. Pretreatment of hepatocytes with either fructose at a concentration of 10mm or NAC at a concentration of 2.5mm prevented 5-MAPB-/MDMA-induced cytotoxicity. In addition, the exposure of hepatocytes to 5-MAPB/MDMA caused the loss of mitochondrial membrane potential, although the preventive effect of fructose was weaker than that of NAC. These results suggest that: (1) 5-MAPB-/MDMA-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were ameliorated, at least in part, by the addition of fructose; and (3) GSH loss via oxidative stress was prevented by NAC. Taken collectively, these results indicate that the onset of toxic effects caused by 5-MAPB/MDMA may be partially attributable to cellular energy stress as well as oxidative stress.

PP88 - Opposite effects of hydrogen sulfide pre- and post-administration on LPS-induced endoplasmic reticulum stress in rat hepatocytes

PP88 - Opposite effects of hydrogen sulfide pre- and post-administration on LPS-induced endoplasmic reticulum stress in rat hepatocytes

PP90 - Hydrogen sulfide preconditioning mitigates LPS-induced endoplasmic reticulum stress in rat hepatocytes through upregulation of HO-1 and opening the ATP-dependent potassium channel

PP90 - Hydrogen sulfide preconditioning mitigates LPS-induced endoplasmic reticulum stress in rat hepatocytes through upregulation of HO-1 and opening the ATP-dependent potassium channel

Endoplasmic reticulum membrane potassium channel dysfunction in high fat diet induced stress in rat hepatocytes.

In a previous study we reported the presence of a large conductance K(+) channel in the membrane of endoplasmic reticulum (ER) from rat hepatocytes. The channel open probability (Po) appeared voltage dependent and reached to a minimum 0.2 at +50 mV. Channel activity in this case was found to be totally inhibited at ATP concentration 2.5 mM, glibenclamide 100 µM and tolbutamide 400 µM. Existing evidence indicates an impairment of endoplasmic reticulum functions in ER stress condition. Because ER potassium channels have been involved in several ER functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the ER potassium channel function is altered in a high fat diet model of ER stress. Male Wistar rats were made ER stress for 2 weeks with a high fat diet. Ion channel incorporation of ER stress model into the bilayer lipid membrane allowed the characterization of K(+) channel. Our results indicate that the channel Po was significantly increased at voltages above +30 mV. Interestingly, addition of ATP 7.5 mM, glibenclamide 400 µM and tolbutamide 2400 µM totally inhibited the channel activities, 3-fold, 4-fold and 6-fold higher than that in the control groups, respectively. Our results thus demonstrate a modification in the ER K(+) channel gating properties and decreased sensitivity to drugs in membrane preparations coming from ER high fat model of ER stress, an effect potentially linked to a change in ER K(+) channel subunits in ER stress condition. Our results may provide new insights into the cellular mechanisms underlying ER dysfunctions in ER stress.

Cytoprotective Effects of Hydrophilic and Lipophilic Extracts of Pistacia vera against Oxidative Versus Carbonyl Stress in Rat Hepatocytes.

This study was conducted to evaluate the cytoprotection of various extracts and bioactive compounds found in Pistacia vera againts cytotoxicity, ROS formation, lipid peroxidation, protein carbonylation, mitochondrial and lysosomal membrane damages in cell toxicity models of diabetes related carbonyl (glyoxal) and oxidative stress (hydroperoxide). Methanol, water and ethyl acetate were used to prepare crude pistachios extracts, which were then used to screen for in-vitro cytoprotection of freshly isolated rat hepatocytes against these toxins. The order of protection by Pistacia vera extracts against both hydroperoxide induced oxidative stress (ROS formation) and glyoxal induced protein carbonylation was: pistachio methanolic extract >pistachio water extract, gallic acid, catechin> α-tochoferol and pistachio ethyl acetate extract. Finally due to higher protection achieved by methanolic extract even compared to sole pretreatment of gallic acid, catechin or α-tochoferol, we suggest that cytoprotection depends on the variety of polar and non-polar compounds found in methanolic extract, it is likely that multiple cytoprotective mechanisms are acting against oxidative and carbonyl induced cytotoxicity. To our knowledge, we are the first to report the cytoprotective activity of Pistacia vera extracts against oxidative and carbonyl stress seen in type 2 diabetes hepatocytes model.

Involvement of increased endogenous asymmetric dimethylarginine in the hepatic endoplasmic reticulum stress of type 2 diabetic rats.

ObjectiveIncreasing evidence suggested that endoplasmic reticulum (ER) stress contributes to insulin resistance, which plays an important role in the development of type 2 diabetes mellitus (T2DM). Accumulation of endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA), is associated with insulin resistance, T2DM, and diabetic cardiovascular complications, although the mechanisms have not been elucidated. This study was to determine whether elevated endogenous ADMA is involved in hepatic ER stress of type 2 diabetic rats, verify their causal relationship, and elucidate the potential mechanism underlying ADMA induced ER stress in rat hepatocytes.MethodsImmunoglobulin binding protein (Bip) transcription, eukaryotic initiation factor 2α kinase (eIF2α) phosphorylation, X box-binding protein-1 (XBP-1) mRNA splicing and C/EBP homologues protein (CHOP) expression were measured to reflect ER stress. Contents of ADMA and nitrite/nitrate as well as activities or expression of NOS and dimethylarginine dimethylaminohydrolase (DDAH) were detected to show the changes in DDAH/ADMA/NOS/NO pathway. The lipid peroxidation product malondialdehyde content and antioxidant enzyme superoxide dismutase activity were analyzed to evaluate oxidative stress.ResultsER stress was provoked in the liver of type 2 diabetic rats, as expressed by increases of Bip transcription, eIF2α phosphorylation, XBP-1 splicing and CHOP expression, all of which were in parallel with the elevation of serum ADMA, suppression of NO generation, NOS and DDAH activities in the liver. Exposure of hepatocytes to ADMA or hydrogen peroxide also induced ER stress, which was associated with the inhibition of NO production and increase of oxidative stress. Treatment of hepatocytes with antioxidant pyrrolidine dithiocarbamate not only decreased ADMA-induced oxidative stress and inhibition of NO production but also reduced ADMA-triggered ER stress.ConclusionsThese results indicate that increased endogenous ADMA contributes to hepatic ER stress in type 2 diabetic rats, and the mechanism underlying ADMA-induced ER stress may relate to oxidative stress via NOS uncoupling.

N-acetylcysteine protects against cadmium-induced oxidative stress in rat hepatocytes

Cadmium (Cd) is a well-known hepatotoxic environmental pollutant. We used rat hepatocytes as a model to study oxidative damage induced by Cd, effects on the antioxidant systems, and the role of N-acetylcysteine (NAC) in protecting cells against Cd toxicity. Hepatocytes were incubated for 12 and 24 h with Cd (2.5, 5, 10 µM). Results showed that Cd can induce cytotoxicity: 10 µM resulted in 36.2% mortality after 12 h and 47.8% after 24 h. Lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase activities increased. Additionally, reactive oxygen species (ROS) generation increased in Cd-treated hepatocytes along with malondialdehyde levels. Glutathione concentrations significantly decreased after treatment with Cd for 12 h but increased after 24 h of Cd exposure. In contrast, glutathione peroxidase activity significantly increased after treatment with Cd for 12 h but decreased after 24 h. superoxide dismutase and catalase activities increased at 12 h and 24 h. glutathione S-transferase and glutathione reductase activities decreased, but not significantly. Rat hepatocytes incubated with NAC and Cd simultaneously had significantly increased viability and decreased Cd-induced ROS generation. Our results suggested that Cd induces ROS generation that leads to oxidative stress. Moreover, NAC protects rat hepatocytes from cytotoxicity associated with Cd.

Protective Effect of Emblica officinalis Against Alcohol-Induced Hepatic Injury by Ameliorating Oxidative Stress in Rats.

The effect of Emblica officinalis fruit extract (EFE) against alcohol-induced hepatic damage in rats was investigated in the present study. In vitro studies showed that EFE possesses antioxidant as well nitric oxide (NO) scavenging activity. In vivo administration of alcohol (5g/kg b.wt/day) for 60days resulted increased liver lipid peroxidation, protein carbonyls, nitrite plus nitrate levels. Alcohol administration also significantly lowers the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and reduced glutathione as compared with control rats. Administration of EFE (250mg/kg body weight) to alcoholic rats significantly brought the plasma enzymes towards near normal level and also significantly reduced the levels of lipid peroxidation, protein carbonyls and restored the enzymic and non-enzymatic antioxidants level. This observation was supplemented by histopathological examination in liver. Our data indicate that the tannoid, flavonoid and NO scavenging compounds present in EFE may offer protection against free radical mediated oxidative stress in rat hepatocytes of animals with alcohol-induced liver injury.

Palmitate protects hepatocytes from oxidative stress and triacylglyceride accumulation by stimulation of nitric oxide synthesis in the presence of high glucose and insulin concentration

Excessive flux of free fatty acids (FFA) into the liver contributes to liver impairment in non-alcoholic fatty liver disease (NAFLD). It remains unclear how FFA contribute to impairment of hepatocytes. This study treated hepatocytes with linoleic acid and palmitate to investigate the early event triggering FFA-mediated impairment. It determined cell viability, content of nitrite/nitrate and triacylglycerides (TG), inducible nitric oxide synthase (iNOS) protein, oxidation of cardiolipin (CL) as well as formation of F2-isoprostanes in the presence of insulin and glucose. Linoleic acid caused significant decrease in cell viability. It is shown that palmitate caused induction of iNOS resulting in increased nitrite/nitrate concentration and slight increase in TG content. Linoleic acid led to a decrease in nitrite/nitrate concentration parallelled by massive TG accumulation in combination with increased oxidation of CL and increased F2-isoprostane levels. It is concluded that nitric oxide (NO) concentration regulates FFA-dependent TG accumulation and oxidative stress in rat hepatocytes.

Ferrylmyoglobin impairs secretion of VLDL triacylglycerols from stored intracellular pools: Involvement of lipid peroxidation

Ferrylmyoglobin (ferrylMb) may play a major role in vivo under certain pathological conditions. Preliminary experiments showed that ferrylmyoglobin induced a mild oxidative stress in rat hepatocytes, mainly reflected by early lipid peroxidation. One of the major functions of hepatocytes is the synthesis, secretion and distribution of lipids to other cells. The aim of this work was to examine whether ferrylMb affected the synthesis and secretion of triacylglycerols (TAG), and the possible involvement of lipid peroxidation on these effects. The heme protein completely impaired VLDL secretion, affecting both the lipid and apoB components of the lipoprotein particle. The incorporation of [ 3H]-oleate into newly synthesized diacylglycerol and TAG was not altered by ferrylMb. The co-treatment of cells with α-tocopherol prevented lipid peroxidation and concomitantly reverted VLDL TAG secretion to control values. Importantly, although ferrylMb dramatically blocked prelabeled TAG secretion, newly synthesized TAG secretion was not impaired. These data indicate that lipid peroxidation elicited by ferrylMb modulates the VLDL TAG secretion process, specifically affecting the stored intracellular TAG mobilization, rather than de novo synthesis. Apart from its potential role in vivo, ferrylmyoglobin constitutes a useful model for studying the interactions between lipid peroxidation and the specific TAG pool dependence for VLDL secretion.

Hepatoprotective Property of Ethanolic and Aqueous Extracts of Fluted Pumpkin (Telfairia occidentalis) Leaves Against Garlic-Induced Oxidative Stress

Fluted pumpkin (Telfairia occidentalis) leaf is a darkish-green leafy vegetable popularly used in soup and in herbal preparations for the management of many diseases in Nigeria. In this study, the hepatoprotective property of ethanolic and aqueous extracts of T. occidentalis leaf (earlier confirmed to have a high level of antioxidant activity) against garlic induced-oxidative stress in rat hepatocytes was investigated. Oxidative stress was induced in Wistar strain albino rats by overdosing them with raw garlic (4%) for 14 days, and this caused a significant increase (P < .05) in serum alkaline phosphatase (ALP), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT), while there was no significant change (P > .05) in serum bilirubin, albumin, globulin, and total proteins. However, intubation of some of the rats fed raw garlic with 5 mg or 10 mg/0.5 mL of T. occidentalis leaf extract (ethanolic or aqueous) caused a significant decrease (P < .05) in serum ALP, GOT, and GPT when compared with rats fed raw garlic without intubation with the T. occidentalis leaf extract. Moreover, 10 mg/0.5 mL of extract was more effective than 5 mg/0.5 mL of extract, while the aqueous extracts appeared to be more effective than the ethanolic extracts in protecting hepatocytes. It could be inferred that both aqueous and ethanolic extracts of T. occidentalis leaf have hepatoprotective properties, although the aqueous extract is more effective than the ethanolic extract, which could be attributed to the higher antioxidant activity of the aqueous extract than the ethanolic extracts of T. occidentalis leaves.

Mechanism of tert-butylhydroperoxide induced apoptosis in rat hepatocytes: involvement of mitochondria and endoplasmic reticulum

The purpose of the present work was to study the mechanisms involved in apoptosis induced by oxidative stress in rat hepatocytes. We focused on the apoptotic signaling molecules cytochrome c, Bcl-2 and Bax. Rat hepatocytes were exposed for 1 h to increasing concentrations of tert-butylhydroperoxide (t-BHP). Using lactate dehydrogenase (LDH) leakage as a biomarker for necrosis, and DNA fragmentation as a biomarker for apoptosis, we observed that a concentration of t-BHP of 0.4–0.5 mM provides a transition point below which apoptosis is favored and beyond which necrosis is favored. Malondialdehyde and 8-oxo-guanine formation indicates that t-BHP induces oxidative stress and damage. However, at 0.4 mM t-BHP, these oxidative molecular changes as well as LDH leakage no longer progress after the first hour of t-BHP exposure, suggesting the activation of some defense mechanisms. Western blot analysis of cytochrome c shows that its level increases in the cytosol while that of Bax decreases in this fraction as a result of t-BHP treatment. Moreover, there is a loss of Bcl-2 from mitochondria while, in contrast, Bax accumulates in this organelle following t-BHP treatment. However, cytochrome c appears to be relocalized to the endoplasmic reticulum as its presence in microsomes is greatly enhanced. We suggest that t-BHP triggers apoptosis through a step that involves cytochrome c release from mitochondria. This event is stimulated by Bcl-2 disappearance from mitochondria and Bax recruitment. Neutralization of excess cytosolic cytochrome c is achieved by its relocalization to the endoplasmic reticulum, hence triggering the down-regulation of apoptotic signals.

Time-dependent oxidative stress caused by benznidazole

Benznidazole (BZN) is a nitroimidazole derivative which has a notable trypanocide activity, and it is the only drug used in Brazil and Argentina for the treatment of Chagas' disease. The drug in current use is thought to act, at least in part, by inducing oxidative stress within the parasite. Imidazolic compounds are involved in the production of reactive oxygen species (ROS). In order to evaluate the effect of BZN on ROS production and on the antioxidant status of the host, male rats were treated for different periods of time (2, 4, 6, 10 and 30 days) with 40 mg BZN/kg body weight. After treatment, biomarkers of oxidative stress such as the activities of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) and glutathione reductase (GR), and also thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), total glutathione (TG) and oxidized glutathione (GSSG) concentrations, were measured in crude hepatic homogenates. Our results revealed that BZN is able to cause tissue damage as shown by increased TBARS content, inhibition of some antioxidants and induction of other antioxidants in a concentration- and time-dependent manner. The tissue damage measured as TBARS increased up to the 10th day of treatment. GST activity was inhibited during the BZN treatment. On the other hand, CAT and GR showed similar increased activities at the beginning, followed by decreased activities at the end of the treatment. After 30 days of treatment, GR activity remained low while CAT activity was high, compared to controls. The SOD activities remained unchanged throughout the experimental period. GSH showed lower values at the beginning of BZN treatment but the hepatic concentrations were enhanced at the end of the experimental period. Total glutathione showed a similar profile, and oxidized glutathione showed higher values in rats treated with BZN. In conclusion, these results indicate that, at therapeutic doses, BZN treatment elicits an oxidative stress in rat hepatocytes.

Activated macrophages increase the susceptibility of rat hepatocytes to ethanol-induced oxidative stress: conflicting effects of nitric oxide.

The aim of this study was to examine how macrophages could act on ethanol-induced oxidative stress in rat hepatocytes during inflammatory conditions, well-known to induce nitric oxide (NO) synthase. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocyte cultures. Co-cultures were then supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN) for 18 h, in order to induce NO synthase before the addition of 50 mM ethanol. In cultures of hepatocytes alone, the addition of LPS and IFN protected from ethanol-induced oxidative stress. It has been shown previously that NO generated in hepatocytes was responsible for this effect. When macrophages were added to primary rat hepatocyte cultures supplemented with LPS and IFN, protection provided by NO against ethanol-induced oxidative stress in hepatocytes ceased. Using a pretreatment of macrophages with N(g)-monomethyl-l-arginine, a NO synthase inhibitor, it was concluded that NO generated by macrophages was responsible for macrophage toxicity. Taken together, our observations suggest that NO biosynthesis in hepatocytes protects them from ethanol-induced oxidative stress, whereas NO production in macrophages deprives hepatocytes of this NO protection.

Effect of nitric oxide on iron-mediated oxidative stress in primary rat hepatocyte culture.

An iron-mediated oxidative stress caused by an increase of the intracellular pool of low molecular weight complex of iron (LMWC) can be observed with iron overloading or ethanol metabolism. The aim of this study was to determine whether nitric oxide (NO) behaved as a pro-oxidant or an antioxidant in such an iron-mediated oxidative stress in rat hepatocytes. The cells were set up in primary cultures and incubated with lipopolysaccharide (LPS) and gamma-interferon (IFN) for 18 hours to induce NO synthase and to trigger NO production. Then 20 micromol/L iron or 50 mmol/L ethanol were added. Oxidative stress was evaluated by measuring lipoperoxidation using two markers: malondialdehyde (MDA) and conjugated dienes. Simultaneously, NO production was followed by the quantitation of nitrites in the culture medium, dinitrosyl iron complexes (DNICs) and mononitrosyl iron complexes (MNICs) in intact hepatocytes. DNIC and MNIC, evaluated by electron paramagnetic resonance (EPR), corresponded to NO bound to iron-containing molecules and to free NO, respectively. In cultures preincubated with LPS and IFN before iron or ethanol addition, a net decrease of lipid peroxidation induced by either NO, iron, or ethanol was noted. Moreover, an elevation of iron-bound NO and a decrease of free NO were observed in these cultures compared with the cultures incubated with only LPS and IFN. These data support the idea that there is a relationship between the changes of NO pool and the inhibition of oxidative stress. In addition, using N(G)-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, NO was shown to be involved in the inhibition of oxidative stress induced by iron or ethanol. Addition of the chelator of LMWC iron, deferiprone, was followed by the inhibition of the increase of iron-bound NO and the reincrease of lipid peroxidation extent, which was as high as in cultures incubated only with LPS and IFN. Thus LMWC iron appeared to be involved also in the inhibition of oxidative stress induced by NO. All the results favor the conclusion that NO acts as an antioxidant in iron-mediated oxidative stress in rat hepatocytes. NO reacted with LMWC iron to form inactive iron complexes unable to induce oxidative stress in rat hepatocytes. Thus NO played a critical role in protecting the liver from oxidative stress.

Effect of nitric oxide on iron-mediated oxidative stress in primary rat hepatocyte culture

An iron-mediated oxidative stress caused by an increase of the intracellular pool of low molecular weight complex of iron (LMWC) can be observed with iron overloading or ethanol metabolism. The aim of this study was to determine whether nitric oxide (NO) behaved as a pro-oxidant or an antioxidant in such an iron-mediated oxidative stress in rat hepatocytes. The cells were set up in primary cultures and incubated with lipopolysaccharide (LPS) and γ-interferon (IFN) for 18 hours to induce NO synthase and to trigger NO production. Then 20 μmol/L iron or 50 mmol/L ethanol were added. Oxidative stress was evaluated by measuring lipoperoxidation using two markers: malondialdehyde (MDA) and conjugated dienes. Simultaneously, NO production was followed by the quantitation of nitrites in the culture medium, dinitrosyl iron complexes (DNICs) and mononitrosyl iron complexes (MNICs) in intact hepatocytes. DNIC and MNIC, evaluated by electron paramagnetic resonance (EPR), corresponded to NO bound to iron-containing molecules and to free NO, respectively. In cultures preincubated with LPS and IFN before iron or ethanol addition, a net decrease of lipid peroxidation induced by either NO, iron, or ethanol was noted. Moreover, an elevation of iron-bound NO and a decrease of free NO were observed in these cultures compared with the cultures incubated with only LPS and IFN. These data support the idea that there is a relationship between the changes of NO pool and the inhibition of oxidative stress. In addition, using NG-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, NO was shown to be involved in the inhibition of oxidative stress induced by iron or ethanol. Addition of the chelator of LMWC iron, deferiprone, was followed by the inhibition of the increase of iron-bound NO and the reincrease of lipid peroxidation extent, which was as high as in cultures incubated only with LPS and IFN. Thus LMWC iron appeared to be involved also in the inhibition of oxidative stress induced by NO. All the results favor the conclusion that NO acts as an antioxidant in iron-mediated oxidative stress in rat hepatocytes. NO reacted with LMWC iron to form inactive iron complexes unable to induce oxidative stress in rat hepatocytes. Thus NO played a critical role in protecting the liver from oxidative stress.(Hepatology 1997 Jan;25(1):122-7)

Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes.

Cell killing, oxygen consumption, and hydroperoxide formation were determined in rat hepatocytes after glycolytic and respiratory inhibition. These conditions model the ATP depletion and reductive stress of anoxia ("chemical hypoxia"). Glycolysis was inhibited with iodoacetate, and mitochondrial electron transfer was blocked with sodium azide, cyanide, or myxothiazol. Cell killing, hydroperoxide formation, and inhibitor-insensitive oxygen consumption were greater after azide than after myxothiazol or cyanide. Desferrioxamine, an inhibitor of iron-catalyzed hydroxyl radical formation, delayed cell killing after each of the respiratory inhibitors. Anoxia also delayed cell killing during chemical hypoxia. However, during anoxic incubations, desferrioxamine did not delay the onset of cell death. These findings indicate that reactive oxygen species participate in lethal cell injury during chemical hypoxia. In isolated mitochondria, previous studies have shown that myxothiazol inhibits Q cycle-mediated ubisemiquinone formation in complex III (ubiquinol-cytochrome c oxidoreductase) and that ubisemiquinone can react with molecular oxygen to form superoxide. Decreased killing of hepatocytes with myxothiazol compared with azide suggests, therefore, that mitochondrial oxygen radical formation by complex III is involved in cell killing during reductive stress. In support of this hypothesis, myxothiazol reduced rates of cell killing and hydroperoxide formation in hepatocytes incubated with azide or cyanide. This mitochondrial mechanism for oxygen radical formation may be important in relative but not absolute hypoxia.

In situ detection of oxidative stress in rat hepatocytes.

In rat hepatocytes in primary culture incubated with nitro blue tetrazolium, formazan content was increased by addition of t-butyl hydroperoxide, a potent oxidant, in a dose-related manner, but not by addition of valinomycin, which kills hepatocytes through mitochondrial damage. This increment after t-butyl hydroperoxide addition was not seen in hepatocytes preincubated with deferoxamine mesylate, a ferric iron chelator which inhibits radical formation. Liver perfusion with nitro blue tetrazolium and t-butyl hydroperoxide in rats produced formazan deposition faintly on the surface of hepatocytes throughout the liver and prominently in the cytoplasm of some hepatocytes, which was attenuated when performed following deferoxamine mesylate perfusion. When liver perfusion with nitro blue tetrazolium was performed in carbon tetrachloride-intoxicated rats, formazan deposition appeared diffusely in hepatocytes in the centrilobular areas. Similar deposition was also observed on the surface and in the cytoplasm of hepatocytes in the periportal and mid-zonal areas in rats undergoing post-ischaemic reperfusion. Liver perfusion with nitro blue tetrazolium can detect in situ oxidative stress in hepatocytes and may be a useful tool for studying the role of lipid peroxidation in rat liver injury.