Oxidative Stress In Liver Cells Research Articles

Protective effects of fermented Rosa roxburghii Tratt juice against ethanol‑induced hepatocyte injury by regulating the NRF2‑AMPK signaling pathway in AML‑12 cells.

Alcohol‑related liver disease (ALD) is a major health concern worldwide. In recent years, there has been growing interest in natural products and functional foods for preventing and treating ALD due to their potential antioxidant and hepatoprotective properties. Rosa roxburghii Tratt, known for its rich content of bioactive compounds, has demonstrated promising health benefits, including anti‑inflammatory and antioxidant effects. Fermentation has been utilized as a strategy to enhance the bioavailability and efficacy of natural products. In the present study, using a mixture of Rosa roxburghii Tratt juice, lotus leaf extract and grape seed proanthocyanidins fermented by Lactobacillus plantarum HH‑LP56, a novel fermented Rosa roxburghii Tratt (FRRT) juice was discovered that can prevent and regulate ethanol‑induced liver cell damage. Following fermentation, the pH was significantly decreased, and the content of VC and superoxide dismutase (SOD) were significantly increased, along with a noticeable enhancement in hydroxyl and 2,2‑diphenyl‑1‑picrylhydrazyl free radical scavenging abilities. Alpha Mouse liver 12 cells were exposed to ethanol for 24 h to establish an in vitro liver cell injury model. The present study evaluated the effects of FRRT on cell damage, lipid accumulation and oxidative stress markers. The results revealed that FRRT pretreatment (cells were pre‑treated with 2.5 and 5 mg/ml FRRT for 2 h) significantly reduced lipid accumulation and oxidative stress in liver cells. Mechanistically, FRRT regulated lipid metabolism by influencing key genes and proteins, such as AMP‑activated protein kinase, sterol regulatory element binding transcription factor 1 and Stearyl‑CoA desaturase‑1. Furthermore, FRRT enhanced antioxidant activity by increasing SOD activity, glutathione and catalase levels, while reducing reactive oxygen species and malondialdehyde levels. It also reversed the expression changes of ethanol‑induced oxidative stress‑related genes and proteins. In conclusion, a novel functional food ingredient may have been discovered with extensive potential applications. These findings indicated that FRRT has antioxidant properties and potential therapeutic benefits in addressing ethanol‑induced liver cell damage through its effects on liver lipid metabolism and oxidative stress.

A microencapsulated blend of botanicals supports weaning piglets during a lipopolysaccharide challenge by modulating liver inflammation and intestinal integrity.

This study examined the action of a blend of botanicals (BOT) against lipopolysaccharide (LPS)-induced inflammation on cultured hepatocytes and weaning piglets. In vitro studies examined HepG2 cells treated with BOT and challenged with Escherichiacoli LPS for 8 d. BOT treatment reduced IL-6 concentration in cell culture media across time (P < 0.05) and decreased pro-inflammatory cytokine expression on days 1 and 8 of experiment (TNFα, IL-1β; P < 0.05). BOT also increased the expression of antioxidant enzymes (GPX-2, SOD, CAT) on day 8 (P < 0.05), which was supported by lowered reactive oxygen species concentration after LPS challenge (P < 0.1). The in vivo study was conducted with 72 weaning pigs, allotted into 24 pens and divided into 3 groups: a negative control (CTR-, basal diet), a challenged control (CTR+) that received an intraperitoneal injection of E. coli O55:B5 LPS on days 14 and 16, and a challenged treated group which received a diet containing 1.5g/kg of microencapsulated BOT (BOT+) for the whole duration of the study. Growth performance was determined weekly and, on days 21 (1 animal per pen) and 28 (remaining animals), pigs were sacrificed to collect liver and jejunal tissues. After the challenge, BOT+ pigs had increased BW on days 21 (P < 0.05) and 28 (P < 0.1) compared to CTR+. Similar improvements in average daily gain and FCR on days 14 to 21 (P < 0.05) and 21 to 28 (P < 0.1) were also seen in BOT+ group. In the liver, compared to CTR+ pigs, BOT+ pigs haddownregulated expression of TLR-4, IL-6, IFN-γ on day 21 (P < 0.05), and TLR-4, TNF-α, IL-8 on day 28 (P < 0.05). BOT+ also increased GPX-2 expression on days 21 and 28 (P < 0.05), while also upregulating SOD-1 and SOD-2 on day 21 (P < 0.05) and CAT on day 28 (P < 0.05) compared to CTR+. In the jejunum, BOT+ reduced inflammation by affecting cytokine expression (P < 0.05) and increasing the expression of tight-junction proteins, ZO-1 on day 21 and CLD-1 on day 28 (P < 0.05). Furthermore, BOT+ pigs had lower crypt depth on days 21 (P < 0.1) and 28 (P < 0.05), and increased villi-to-crypt ratio on days 21 and 28 (P < 0.05). By day 28, BOT+ intestinal measurements were restored to values similar to the CTR-. Finally, BOT+ also reduced mast cell activation on day 21 (P < 0.05) compared to CTR+. Considering all the findings, BOT controlled inflammatory activation and oxidative stress in liver cells, enhanced intestinal integrity, and as a result improved the growth performance of weaning piglets challenged with LPS.

Modelling porcine NAFLD by deletion of leptin and defining the role of AMPK in hepatic fibrosis

BackgroundNon-alcoholic fatty liver disease (NAFLD) is the most prevalent cause of chronic hepatic disease and results in non-alcoholic steatohepatitis (NASH), which progresses to fibrosis and cirrhosis. Although the Leptin deficient rodent models are widely used in study of metabolic syndrome and obesity, they fail to develop liver injuries as in patients.MethodsDue to the high similarity with humans, we generated Leptin-deficient (Leptin−/−) pigs to investigate the mechanisms and clinical trials of obesity and NAFLD caused by Leptin.ResultsThe Leptin−/− pigs showed increased body fat and significant insulin resistance at the age of 12 months. Moreover, Leptin−/− pig developed fatty liver, non-alcoholic steatohepatitis and hepatic fibrosis with age. Absence of Leptin in pig reduced the phosphorylation of JAK2-STAT3 and AMPK. The inactivation of JAK2-STAT3 and AMPK enhanced fatty acid β-oxidation and leaded to mitochondrial autophagy respectively, and both contributed to increased oxidative stress in liver cells. In contrast with Leptin−/− pig, although Leptin deletion in rat liver inhibited JAK2-STAT3 phosphorylation, the activation of AMPK pathway might prevent liver injury. Therefore, β-oxidation, mitochondrial autophagy and hepatic fibrosis did not occurred in Leptin−/− rat livers.ConclusionsThe Leptin-deficient pigs presents an ideal model to illustrate the full spectrum of human NAFLD. The activity of AMPK signaling pathway suggests a potential target to develop new strategy for the diagnosis and treatment of NAFLD.

Regulatory effect of PINK1/Parkin axis on mitophagy in isoniazide-induced hepatocellular injury.

Patients with tuberculosis, who are treated with long-term high-dose combined use of anti-tuberculosis drugs, can cause many adverse reactions such as liver damage, but the mechanism is still unclear. Although phosphatase and tensin homolog induced kinase 1 (PINK1)/Parkin axis might participate in the process of liver damage through regulating mitochondrial autophagy and oxidative stress in liver cells. However, the association between the mitochondrial autophagy regulated by the PINK1/Parkin axis and the liver injury induced by anti-tuberculosis drugs is unknown. This study aims to explore the mechanism of PINK1/Parkin signal axis in regulating hepatocellular injury induced by anti-tuberculosis drugs through mitochondrial autophagy, and to provide new therapeutic targets for the patients with liver damage caused by anti-tuberculosis drugs. Mouse hepatocytes AML-12 were treated with isoniazide (INH) to induce liver injury. The mRNA and protein levels of PINK1, Parkin and autophagy associated factors were detected by real-time PCR and Western blotting in the normal AML-12 cells (control group), the AML-12 cells treated by INH (model group) and the AML-12 cells treated with INH and salidroside together (intervention group). Meantime, ELISA kit was used to detect the level of reactive oxygen species (ROS) in AML-12 cells, and the cell morphology and injury was observed by HE staining and transmission electron microscope (TEM). Compared with the control group, the mRNA (all P <0.01) and protein levels (all P <0.05) of PINK1, Parkin and microtubule associated protein 1 light chain 3 (LC3) were significantly decreased, the ubiquitination level was significantly decreased ( P <0.05), and the ROS level was increased ( P <0.05), and the hepatocellular cell showed obvious damage in the model group. Compared with the model group, the expression of PINK1, Parkin and LC3 were significantly increased in the intervention group (all P <0.05), as well as the ubiquitination level was also increased ( P <0.05). The ROS level mediated by mitochondria was decreased ( P <0.05). The results of HE staining and TEM showed that the cell status was improved and the damage of hepatocytes was significantly attenuated. In this study, the mechanism of mitochondrial autophagy mediated by PINK1/Parkin signal axis may be related to INH-induced injury in liver cells and it can regulate the damage state in vitro, indicating that Parkin is a potential molecular target for the prediction, diagnosis and treatment for liver injury induced by anti-tuberculosis drugs.

Protective action of polysaccharides from Laurencia papillose (Rhodophyta) against imidacloprid induced genotoxicity and oxidative stress in male albino rats.

Imidacloprid (IMI), the main component of neonicotinoid insecticides, promotes oxidative stress and genotoxicity in mammals. The aim of this experiment is to assess oxidative stress in liver cells and genotoxicity of erythrocytes for rats exposed to sub-lethal doses of IMI and the protective effects for Rhodophyta as antioxidant material versus imidacloprid. A total of 30 adult male albino rats (average body weight, 190–200 g) were divided into six groups (n=5) as follows: group 1 served as the control, group 2 received 200 mg/kg red algae, group 3 received 45 mg/kg IMI (high-dose group), group 4 received 22.5 mg/kg IMI (low-dose group), group 5 received 200 mg/kg red algae +45 mg/kg IMI, and group 6 received 200 mg/kg red algae +22.5 mg/kg IMI. After 28 d of treatment, the antioxidant activity of the crude extract of red algae was assessed in terms of free radical scavenging activity and found to be higher in TCA (75.57%) followed by DPPH (50.08%) at concentration 100 μg extract and a significant increase in lipid peroxidation and reductions in glutathione were observed in liver cells were intoxicated with high and low doses of IMI. Moreover decreases in catalase and glutathione peroxidase parameters in same previous groups which indicated oxidative stress. In addition significant increases in micronucleus frequency (MN) in the bone marrow of the rats as a genotoxicity marker which indicated DNA damage in erythrocytes cells with alterations in the histopathology of liver cells were also noted such as necrosis, inflammatory cells, infiltration, and necrobiotic changes. Whereas Rhodophyta succeeded in alleviation the oxidative damage and genotoxicity induced by the insecticide. In conclusion, IMI demonstrates hazardous effects, such as alterations in antioxidant status and mutagenicity of erythrocytes and polysaccharides from Rhodophyta has good antioxidant activity in vivo model systems against imidacloprid.

High-Content Screening for the Detection of Drug-Induced Oxidative Stress in Liver Cells.

Drug-induced liver injury (DILI) remains a major cause of drug development failure, post-marketing warnings and restriction of use. An improved understanding of the mechanisms underlying DILI is required for better drug design and development. Enhanced reactive oxygen species (ROS) levels may cause a wide spectrum of oxidative damage, which has been described as a major mechanism implicated in DILI. Several cell-based assays have been developed as in vitro tools for early safety risk assessments. Among them, high-content screening technology has been used for the identification of modes of action, the determination of the level of injury and the discovery of predictive biomarkers for the safety assessment of compounds. In this paper, we review the value of in vitro high-content screening studies and evaluate how to assess oxidative stress induced by drugs in hepatic cells, demonstrating the detection of pre-lethal mechanisms of DILI as a powerful tool in human toxicology.

Adiponectin alleviates non-alcoholic fatty liver injury via regulating oxidative stress in liver cells.

The aim of the present study was to investigate the role of adiponectin in non-alcoholic fatty liver cell model and its mechanism. The serum were collected from patients with non-alcoholic fatty liver disease and healthy controls. Then the expression of APN in the serum was detected using APN kit. Furthermore, an in vitro model of NAFLD was established using mixed fatty acids treated HepG2 cells, and APN was highly expressed in the culture solution to a concentration of 10 μg/mL. The normal control group (Normal) was normal cells, the model group (NAFLD) was mixed fatty acids treated HepG2 cells, the experimental group (NAFLD+APN) was model cells transfected with high APN expression, and the negative control group (NAFLD+PBS) was model cells transfected with PBS. The expression of NOX2 in each group was detected by Western blot. The corresponding kit was used to detect the level of triglyceride (TG), the activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA), and the ratio of GSH/GSSG in each group of cells. The expression level of APN was greatly decreased in the serum of NAFLD patients (P<0.01), and the TG content was significantly increased in HepG2 cells treated with fatty acids (P<0.001), indicating successful modeling. The cells had high expression of APN (P<0.001) showed low expression of NOX2 (P<0.001). The kit test results showed that the high expression of APN could reverse the decrease of SOD activity, the increase of MDA level, the decrease of GSH/GSSG ratio and the increase of TG content (P<0.001), all of which were restored to the modeling level after application of NOX2's activator TBCA. APN was lowly expressed in the serum of NAFLD patients. Its effect mechanism was to alleviate the injury of NAFLD cells by reducing oxidative stress via regulating NOX2 expression.

Rosmarinic Acid Ameliorates H2O2-Induced Oxidative Stress in L02 Cells Through MAPK and Nrf2 Pathways.

Liver cells are easily damaged by oxidative stress during progression both in liver development and throughout adult life, resulting in tissue pathology that ranges from simple hepatitis to nonalcoholic fatty liver disease. In this study, we determined the attenuation of oxidative stress in liver cells with pretreatment of rosmarinic acid (RA), which is an antioxidant agent from Rosmarinus officinalis. The human liver cell line L02 was damaged by hydrogen peroxide (H2O2). In the RA treatment group, the viability of L02 cells increased and the intracellular reactive oxygen species levels decreased compared with the H2O2-induced damage group. Analysis of flow cytometry revealed that the percentage of G2/M cell cycle arrest and cell apoptosis decreased in the RA treatment group. This alteration was associated with activation of a G2/M DNA damage and oxidative stress apoptotic signal. Furthermore, we determined the redox-sensitive protein expression of mitogen-activated protein kinases (MAPKs), quinone acceptor oxidoreductase 1 (NQO1), and nuclear factor E2-related factor 2 (Nrf2), and the expression of both MAPKs and Nrf2 was activated in the RA group. Results showed that the relevant protein expression of MAPKs and Nrf2 was activated in the RA group. Thus, RA protected L02 cells from oxidative damage through suppressing cell cycle arrest and cell apoptosis with the activation of MAPK and Nrf2 signaling pathways.

Liver enzyme trends in patients taking uninterrupted metformin before and after coronary surgery.

Metformin is an oral antidiabetic agent belonging to the biguanide class of antidiabetics. Possible novel applications for metformin in cardiovascular disease might exist. The aim of this study was to verify a possible association between pre-operative metformin administration and protection against ischemia-induced liver injury in diabetic patients undergoing coronary artery bypass grafting (CABG) surgery. A retrospective case-control series was conducted at a single center. Two hundred consecutive diabetic patients underwent isolated on-pump CABG during a 12-month span (July 2015 - July 2016). Metformin was uninterrupted in patients who took this drug prior to CABG; 68 patients were metformin users (34%) while 132 patients were taking other antidiabetic agents (66%). Liver enzymes and other organ markers were consecutively recorded daily for 7 days after surgery and expressed as medians with interquartile range (IQR). Both the metformin and non-metformin group of patients had similar pre-operative demographic characteristics. The median (IQR) post-operative cardiac enzyme creatinine kinase (CK) MB fraction was significantly lower in the metformin group [46.4 U/L (35.8-66.5) vs. 66.5 U/L (44-94.5), P=0.005]. Total bilirubin [0.58 (0.48-0.82) mg/dL vs. 0.67 (0.56-0.95) mg/dL, P=0.021], the transaminase aspartate aminotransferase (AST) [32.5 U/L (25.0-42.0) vs. 37.5 U/L (28.5-56), P=0.011], the transaminases alanine aminotransferase (ALT) [48.5 U/L (40.0-64.0) vs. 57.0 U/L (44.0-77.0), P=0.040] and lactate dehydrogenase (LDH) [320.0 U/L (273.5-367.2) vs. 356.5 U/L (289.5-427), P=0.014] were significantly lower in the metformin group. No differences were noted in clinical outcomes. In this limited retrospective study, the diabetic patients who took metformin before and after undergoing CABG appeared to have a reduced post-operative surge in the total bilirubin and transaminase liver enzymes. Metformin's role in mitigating oxidative stress in liver cells might explain this observation. Further experimental studies are warranted to verify this possible effect.

Oleuropein-Rich Olive Leaf Extracts May Ameliorate Consequences of Glucose-Induced Oxidative Stress in Hep G2 Cells

Olive leaf is reputed to have many beneficial health-related properties, often attributed to the iridoid glycoside oleuropein. In this work, the composition and antioxidant activity of oleuropein-rich olive leaf extracts prepared by ultrasonication were investigated. In addition, the influence of the extracts on Hep G2 cells subjected to glucose-induced oxidative stress was examined. The prepared extracts acted as antioxidants rich in oleuropein and other polyphenols. Among them, the extract prepared at 20°C using 100% ethanol was the most effective radical scavenger, antioxidant and metal chelator. It also contained the largest amount of oleuropein and other phenolic constituents. The extract prepared at 20°C using 70% ethanol, on the other hand, showed the best yield among the investigated extracts. Furthermore, it was able to preserve the cell-membrane integrity of Hep G2 cells subjected to glucose-induced oxidative stress, as well as to maintain intracellular concentration of glutathione and the activity of glutathione S-transferase on the levels comparable to those observed in non-stressed cells. The presented results demonstrate that oleuropein-rich olive leaf extracts can effectively ameliorate the consequences of glucose-induced oxidative stress in liver cells.

Protective role of biliverdin against bile acid-induced oxidative stress in liver cells

The accumulation of bile acids affects mitochondria causing oxidative stress. Antioxidant defense is accepted to include biotransformation of biliverdin (BV) into bilirubin (BR) through BV reductase α (BVRα). The mutation (c.214C>A) in BLVRA results in a non-functional enzyme (mutBVRα). Consequently, homozygous carriers suffering from cholestasis develop green jaundice. Whether BVRα deficiency reduces BV-dependent protection against bile acids is a relevant question because a screening of the mut-BLVRA allele (a) in 311 individuals in Greenland revealed that this SNP was relatively frequent in the Inuit population studied (1% a/a and 4.5% A/a). In three human liver cell lines an inverse correlation between BVRα expression (HepG2>Alexander>HuH-7) and basal reactive oxygen species (ROS) levels was found, however the ability of BV to reduce oxidative stress and cell death induced by deoxycholic acid (DCA) or potassium dichromate (PDC) was similar in these cells. The transduction of BVRα or mutBVRα in human placenta JAr cells with negligible BVRα expression or the silencing of endogenous BVRα expression in liver cells had no effect on DCA-induced oxidative stress and cell death or BV-mediated cytoprotection. DCA stimulated both superoxide anion and hydrogen peroxide production, whereas BV only inhibited the latter. DCA and other dihydroxy-bile acids, but not PDC, induced up-regulation of both BVRα and heme oxygenase-1 (HO-1) in liver cells through a FXR independent and BV insensitive mechanism. In conclusion, BV exerts direct and BVRα-independent antioxidant and cytoprotective effects, whereas bile acid accumulation in cholestasis stimulates the expression of enzymes favoring the heme biotransformation into BV and BR.

High hydrostatic pressure influences the in vitro response to xenobiotics in Dicentrarchus labrax liver

Hydrostatic pressure (HP) increases by about 1 atmosphere (0.1MPa) for each ten-meter depth increase in the water column. This thermodynamical parameter could well influence the response to and effects of xenobiotics in the deep-sea biota, but this possibility remains largely overlooked. To grasp the extent of HP adaptation in deep-sea fish, comparative studies with living cells of surface species exposed to chemicals at high HP are required. We initially conducted experiments with precision-cut liver slices of a deep-sea fish (Coryphaenoides rupestris), co-exposed for 15h to the aryl hydrocarbon receptor (AhR) agonist 3-methylcholanthrene at HP levels representative of the surface (0.1MPa) and deep-sea (5–15MPa; i.e., 500–1500m depth) environments. The transcript levels of a suite of stress-responsive genes, such as the AhR battery CYP1A, were subsequently measured (Lemaire et al., 2012; Environ. Sci. Technol. 46, 10310–10316). Strikingly, the AhR agonist-mediated increase of CYP1A mRNA content was pressure-dependently reduced in C. rupestris. Here, the same co-exposure scenario was applied for 6 or 15h to liver slices of a surface fish, Dicentrarchus labrax, a coastal species presumably not adapted to high HP. Precision-cut liver slices of D. labrax were also used in 1h co-exposure studies with the pro-oxidant tert-butylhydroperoxide (tBHP) as to investigate the pressure-dependence of the oxidative stress response (i.e., reactive oxygen production, glutathione and lipid peroxidation status). Liver cells remained viable in all experiments (adenosine triphosphate content). High HP precluded the AhR agonist-mediated increase of CYP1A mRNA expression in D. labrax, as well as that of glutathione peroxidase, and significantly reduced that of heat shock protein 70. High HP (1h) also tended per se to increase the level of oxidative stress in liver cells of the surface fish. Trends to an increased resistance to tBHP were also noted. Whether the latter observation truly reflects a protective response to oxidative stress will be addressed in future co-exposure studies with both surface and deep-sea fish liver cells, using additional pro-oxidant chemicals. Altogether, data on CYP1A inducibility with D. labrax and C. rupestris support the view that high HP represses AhR signaling in marine fishes, and that only species adapted to thrive in the deep-sea have evolved the molecular adaptations necessary to counteract to some extent this inhibition.

Quercetin Protects Liver Cells and Mitochondria Against Doxorubicin Induced Oxidative Stress in Albinos' Rats

Doxorubicin (DOX) is considered the most toxic anthracycline on hepatocytes. Despite this toxicity, DOX is widely used in clinical oncology practice due to its good therapeutic efficacy. Considerable effort has been expended to identify therapies that reduce this adverse response. This study treats the preventive effect of Quercetin (QE); one of the most abundant bioflavonoids, against doxorubicin induced oxidative stress in liver cells and mitochondria. We find that doxorubicin at the amount of 10 mg/Kg altered liver mitochondrial functions as attested by the overproduction of superoxide anion production (O2–) by mitochondrial respiratory chain complex III. The hepatic tissue from doxorubicin treated rats showed a marked depletion in reduced GSH content, a significant increase in malonedialdehyde (MDA) levels and inhibition in superoxide dismutase (Mn-SOD), (Cu-Zn SOD) and catalase (CAT) enzymatic activities. These results are reversed after one mount per os pretreatment by Quercetin at the amount of 0.33 mg/kg. Quercetin protects liver tissue from oxidative stress by protecting mitochondria and reinforcement of enzymatic and non enzymatic antioxidant defenses.

Glucagon-like peptide-1: New target for treating non-alcoholic fatty liver disease?

Recent studies have demonstrated an association of glucagon-like peptide-1(GLP-1)signaling defect with non-alcoholic fatty liver disease(NAFLD). Native GLP-1 and GLP-1-based agents may directly act on hepatocytes through activation of GLP-1 receptors in hepatocytes, resulting in the regulation of gene expression associated with insulin resistance and lipid metabolism, and the suppression of oxidative stress in liver cells. Moreover, GLP-1-based agents have been reported to be effective in improving hepatic endpoints in patients with NAFLD. (Chin J Endocrinol Metab, 2015, 31: 386-389) Key words: Non-alcoholic fatty liver disease; Glucagon-like peptide-1; Dipeptidyl peptidase-4

In Vitro Assessment of Cadmium Bioavailability in Chinese Cabbage Grown on Different Soils and Its Toxic Effects on Human Health.

The minimum concentration of cadmium (Cd), by Chinese cabbage grown on Cd contaminated soils that can initiate toxicity in human liver cells using in vitro digestion coupled with Caco-2/HL-7702 cell models was studied. Cadmium bioaccessibility in the gastric phase for yellow soil (YS) cabbage (40.84%) and calcareous soil (CS) cabbage (21.54%) was significantly higher than small intestinal phase with the corresponding values of 21.2% and 11.11%, respectively. Cadmium bioavailability was higher in YS cabbage (5.27%–14.66%) than in CS cabbage (1.12%–9.64%). Cadmium concentrations (>0.74 μg) transported from YS and CS cabbage were able to induce oxidative (MDA, H2O2) stress by inhibiting antioxidant (SOD, GPx) enzyme activities in human liver cells (HL-7702). Additionally the study revealed that the ingestion of Cd contaminated Chinese cabbage grown in acidic soil (yellow soil) weakened the antioxidant defense system under all levels of contamination (2, 6, and 9 mg·kg−1) which ultimately escalated the oxidative stress in liver cells; however, in case of CS cabbage, a marked oxidative stress was observed only at 9 mg kg−1 Cd level of soil. Therefore, it is necessary to monitor Cd concentrations in leafy vegetables grown on acidic soils to minimize human health risk.

Establishment of the Metabolite Profile for an Antrodia cinnamomea Health Food Product and Investigation of Its Chemoprevention Activity

Antrodia cinnamomea is an edible fungus endemic to Taiwan that has been attributed with health promotion benefits. An A. cinnamomea mycelium health food product, which was produced by solid-state culture, was selected as the target for investigation in this study. Fourteen representative metabolites of A. cinnamomea mycelium (EMAC) were selected as index compounds to establish the metabolite profile for evaluation of EMAC product quality. It was also demonstrated that EMAC administration significantly reduced liver inflammation and serum oxidative stress in vivo. 4-Acetylantroquinonol B obtained by a bioactivity-guided fractionation from EMAC was able to not only inhibit LPS-induced nitric oxide formation in macrophages but also protect against ethanol-induced oxidative stress in liver cells. The results suggest this A. cinnamomea product might be a potent antioxidative and anti-inflammatory supplement for chemoprevention.

MiR-214 Promotes the Alcohol-Induced Oxidative Stress via Down-Regulation of Glutathione Reductase and Cytochrome P450 Oxidoreductase in Liver Cells

The involvement of oxidative stress in the pathophysiological process of alcohol-induced liver injury has been studied for decades. However, the role of microRNAs (miRNAs) targeting to oxidative stress genes in the pathogenesis of alcohol-induced liver injury has not yet been determined. The aim of this study was to identify the targeting of miR-214 to both glutathione reductase (GSR) and cytochrome P450 oxidoreductase (POR) genes and elucidate their impact on alcohol-induced oxidative stress in liver cells. The miR-214 expression vector and reporter vectors of GSR and POR 3'-UTR were constructed. Human hepatoma cell (Bel7402), human embryonic kidney 293 cell (HEK293), and rat normal hepatocyte (BRL) were transfected and stimulated with ethanol (EtOH). Wistar rats were fed with EtOH for 4 weeks. The GSR and POR protein levels were detected by Western blot, and their activities were measured using the spectrophotometric method. The miR-214 expression was detected by real-time PCR. The index of oxidative stress including the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) level was detected by commercial kits. miR-214 bound specifically to the GSR and POR 3'-UTR and repressed the expressions and activities of both GSR and POR. EtOH up-regulated the miR-214 expression, down-regulated the GSR and POR protein levels and activities, and induced the oxidative stress in human and rat liver cells. EtOH-fed Wistar rats further confirmed that alcohol up-regulates the miR-214 expression in liver and repressed both GSR and POR in vivo. These findings demonstrated a new mechanism by which the alcohol repressed the GSR and POR expression via up-regulation of miR-214 and in turn induced oxidative stress in liver cells.

Evaluation of epirubicin-induced acute oxidative stress toxicity in rat liver cells and mitochondria, and the prevention of toxicity through quercetin administration

Anticancer therapy with epirubicin (EPI) results in acute hepatotoxicity, likely due to the generation of free radicals. However, the oxidative status of rat liver cells and mitochondria after EPI toxicity has not been investigated. In the present study, we first investigated the pro-oxidant effect of EPI on both hepatic cells and mitochondrial function. Injection of EPI into rats at a dose of 9 mg/kg (cumulative dose in human chemotherapy), induced hepatic dysfunction, as revealed by a significant increase in serum glutamate oxaloacetate transaminases (SGOT) and glutamate pyruvate transaminases (SGPT). Oxidative stress in liver cells and mitochondria was provoked by EPI because a statistically significant reduction of catalase (CAT), superoxide dismutase (SOD) and cytosolic glutathione (GSH) levels, and a significant increase in malonedialdehyde (MDA) levels – an indicator of lipid peroxidation that can perforate biological membranes – were observed. Second, the protective effect of quercetin (QE) (0.33 mg/kg) against EPI-induced oxidative stress was also investigated. Indeed, the pretreatment of rats with QE protected liver cells and mitochondria from oxidative stress. This treatment prevented hepatic dysfunction by maintaining normal levels of serum transaminases following the inhibition of their hepatic leakage by preventing lipid peroxidation. Thus, QE works through the prevention of cellular membrane perforation and the antioxidant defense system of mitochondria from liver cells, which represent compartments for the permanent production of reactive oxygen species (ROS) through the respiratory chain.

Transcriptional profiling of mouse liver in response to chronic heat stress

Mice were used to study the effects of chronic heat stress on hepatic gene expression. Twenty-five mice were allocated to either chronic heat stress (34 °C) or control (24 °C) conditions for a period of 2 weeks from 47 to 60 d of age. Nineteen genes differentially expressed in liver were identified using DNA microarrays. Genes involved in the anti-oxidant pathway and metabolism were up-regulated. Genes involved in generation of reactive oxygen radicals and mitochondrial expressed genes were down-regulated. Enzyme activity measurements confirmed the array results. Mice exposed to chronic heat stress showed signs of increased oxidative stress in liver cells.

Hydrogen peroxide generation in peroxisome proliferator-induced oncogenesis

Peroxisome proliferators are a structurally diverse group of non-genotoxic chemicals that induce predictable pleiotropic responses including the development of liver tumors in rats and mice. These chemicals interact variably with peroxisome proliferator-activated receptors (PPARs), which are members of the nuclear receptor superfamily. Evidence derived from mice with PPARα gene disruption indicates that of the three PPAR isoforms (α, β/δ and γ), the isoform PPARα is essential for the pleiotropic responses induced by peroxisome proliferators. Peroxisome proliferator-induced activation of PPARα leads to profound transcriptional activation of genes encoding for the classical peroxisomal β-oxidation system and cytochrome P450 CYP 4A isoforms, CYP4A1 and CYP4A3, among others. Livers with peroxisome proliferation manifest substantial increases in the expression of H 2O 2-generating peroxisomal fatty acyl-CoA oxidase, the first enzyme of the classical peroxisomal fatty acid β-oxidation system, and of microsomal cytochrome P450 4A1 and 4A3 genes. Disproportionate increases in H 2O 2-generating enzymes and H 2O 2-degrading enzyme catalase and reductions in glutathione peroxidase activity by peroxisome proliferators, lead to increased oxidative stress in liver cells. Sustained oxidative stress resulting from chronic increases in H 2O 2-generating enzymes manifests as massive accumulation of lipofuscin in hepatocytes, and increased levels of 8-hydroxydeoxyguanosine adducts in liver DNA; this supports the hypothesis that oxidative stress plays a critical role in the development of liver tumors induced by these non-genotoxic chemical carcinogens. Evidence also indicates that cells stably overexpressing H 2O 2-generating fatty acyl-CoA oxidase or urate oxidase, when exposed to appropriate substrate(s), reveal features of neoplastic conversion including growth in soft agar and formation of tumors in nude mice. Mice with disrupted fatty acyl-CoA oxidase gene (AOX −/− mice), which encodes the first enzyme of the PPARα regulated peroxisomal β-oxidation system, exhibit profound spontaneous peroxisome proliferation, including development of liver tumors, indicative of sustained activation of PPARα by the unmetabolized substrates of acyl-CoA oxidase. With the exception of fatty acyl-CoA oxidase, all PPARα responsive genes including CYP4A1 and CYP4A3 are up-regulated in the livers of these AOX −/− mice. Thus, the substrates of acyl-CoA oxidase serve as endogenous ligands for this receptor leading to a receptor–enzyme cross-talk, because acyl-CoA oxidase gene is transcriptionally regulated by PPARα. Peroxisome proliferators induce only a transient increase in liver cell proliferation and this may serve as an additional contributory factor, rather than play a primary role in liver tumor development. Thus, sustained activation of PPARα by either synthetic or natural ligands leads to reproducible pleiotropic responses culminating in the development of liver tumors. This phenomenon of peroxisome proliferation provides fascinating challenges in exploring the molecular mechanisms of cell specific transcription, and in identifying the PPARα responsive target genes, as well as events involved in their regulation. Genetically altered animals and cell lines should enable investigations on the role of H 2O 2-producing enzymes in neoplastic conversion.