The effect of silymarin (Silybum marianum) on human skin fibroblasts in an in vitro wound healing model

Context: Silymarin, a flavonolignan from Silybum marianum (L.) Gaertn. (Asteraceae), has been reported to have antioxidant and anti-inflammatory properties. Therefore, it may be worthwhile to study the effect of silymarin on wound healing.

Objective: To evaluate the effect of silymarin on human fibroblast cells in an in vitro model of wound healing.

Materials and methods: Human fibroblast cells were treated with different concentrations (4.5, 9, 18, 36 µg/mL) of silymarin. The effects of silymarin on cell viability, proliferation, collagen synthesis, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2′-deoxy-uridine, hydroxyproline analysis and real-time PCR, respectively. The effect of silymarin on cellular antioxidant status was determined by protection against hydrogen peroxide (H₂O₂)-induced cell injury and free radical scavenging activity (ABTS assay) of the cells.

Results: Results of the present study indicate that pretreatment of fibroblast cells with silymarin significantly protected cells against H₂O₂-induced injury (p < 0.05). After an 18?h treatment of cells with 36 µg/mL silymarin, total antioxidant capacity of cells significantly increased (p < 0.05). Furthermore, pretreatment of human fibroblast cells with silymarin significantly inhibited lipopolysaccharide (LPS)-induced COX-2 mRNA expression (p < 0.001). There was no significant difference in fibroblast proliferation and collagen synthesis between treatment and control groups (p > 0.05).

Discussion and conclusion: Silymarin may be useful as a therapeutic agent for the treatment of cutaneous wounds through its antioxidation and anti-inflammation effects.     

Tissue-specific effects of fenthion on glutathione metabolism modulated by NAC and BSO in Oreochromis niloticus

Introduction:?The present study was designed to understand the effects of organophosphate (OP) insecticide and avicide fenthion on cellular redox status and the role of reduced glutathione (GSH) on fenthion toxicity in the liver and kidney of Oreochromis niloticus as a model organism. N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) were injected intraperitoneally to fenthion-exposed fish as modulators of GSH metabolism. GSH redox status, GSH-related enzyme activities, and thiobarbituric acid reactive substances (TBARS) contents were then measured spectrophotometrically at 24, 48, and 96 hours. To assess recovery from fenthion exposure, similar analyses were performed on fish transferred to non-treated water for 24, 48, and 96 hours.

Results:?Fenthion increased glutathione S-transferase (GST; EC 2.5.1.18) activity and caused changes in total GSH (tGSH), GSH and oxidized glutathione (GSSG) contents and glutathione peroxidase (GPx; EC 1.11.1.9) specific activity in the liver tissue over time. Increases observed in tGSH and GSSG contents at 24 hours were decreased by fenthion treatment at 96 hours. BSO caused a sharp decline in liver tGSH, GSH, and GSSG contents and an elevation in GST and γ-glutamyl transpeptidase (γ-GT; EC 2.3.2.2) enzyme activities. A significant decrease was observed in tGSH and GSH contents and, also, GST enzyme activities in the kidney at 48-hour fenthion treatment. On the contrary to the liver, a significant increase was observed in tGSH and GSH contents in the kidney by BSO injection. NAC application eliminated the decreasing effects of fenthion on GST activity in this tissue. NAC injection caused decreases in lipid peroxidation (LPO) levels. Decline in tGSH and GSH contents were maintained in the liver during the recovery period, and elevations in LPO levels in the kidney were observed during the same period.

Conclusions:?In conclusion, tissue-specific and time-dependent GSH redox status disturbance of fenthion were observed. BSO revealed the significance of GST-mediated GSH conjugation on the detoxification process of fenthion. NAC seemed useful to avoid the fenthion-related oxidative toxicity.     

Structural isomerization of synephrine influences its uptake and ensuing glutathione depletion in rat-isolated cardiomyocytes

Synephrine is a natural compound, frequently added to ephedra-free dietary supplements for weight-loss, due to its effects as a nonspecific adrenergic agonist. Though only p-synephrine has been documented in plants, the presence of m-synephrine has also been reported in weight-loss products. The use of synephrine in dietary supplements was accompanied by reports of adverse effects, especially at the cardiovascular level. It is well known that the imbalance in cardiac glutathione levels can increase the risk of cardiomyopathy. The present work aimed to study the role of organic cation-mediated transport of m- and p-synephrine and the possibility that p- and m-synephrine induce intracellular changes in glutathione levels in calcium-tolerant freshly isolated cardiomyocytes from adult rat. After a 3 h incubation with 1 mM p- or m-synephrine, the intracellular content of synephrine was measured by gas chromatography/ion trap-mass spectrometry (GC/IT-MS); cell viability and intracellular glutathione levels were also determined. To evaluate the potential protective effects of antioxidants against the adverse effects elicited by m-synephrine, cells were pre-incubated for 30 min with Tiron (100 μM) or N-acetyl-cysteine (NAC) (1 mM). To assess the influence of α₁-adrenoceptors activation in glutathione depletion, a study with prazosin (100 nM) was also performed. The results obtained provide evidence that organic cation transporters OCT3 and OCT1 play a major role in m- and p-synephrine-mediated transport into the cardiomyocytes. The importance of these transporters seems similar for both isomers, although p-synephrine enters more into the cardiomyocytes. Furthermore, only m-synephrine induced intracellular total glutathione (GSHt) and reduced glutathione (GSH) depletion. NAC and Tiron were able to counteract the m-synephrine-induced GSH and GSHt decrease. On the other hand, the incubation with prazosin was not able to change m-synephrine-induced glutathione depletion showing that this effect is independent of α₁-adrenoceptor stimulation. In conclusion, both positional isomers require OCT3 and OCT1-mediated transport to enter into the cardiomyocytes; however, the hydroxyl group in the p-position favours the OCT-mediated transport into cardiomyocytes. Furthermore, the structural isomerization of synephrine influences its toxicological profile since only m-synephrine caused GSH depletion.     

Naringenin attenuates CCl4‐induced hepatic inflammation by the activation of an Nrf2‐mediated pathway in rats

The possible protective effects of naringenin, a naturally occurring citrus flavonone, on carbon tetrachloride (CCl₄)‐induced liver injury in rats and the mechanism underlying its effects were investigated. Forty rats were divided into five groups. Rats in Groups I and II served as the normal and injured liver groups, respectively; Group III rats were treated with the standard drug silymarin as a positive control; and rats in Groups IV and V (naringenin‐treated groups) were administrated 50 mg/kg, p.o., naringenin for 7 days. Liver samples were collected to evaluate mRNA and protein expression, histological changes and oxidative stress. Naringenin inhibited lipid peroxidation and reduced serum levels of hepatic enzymes induced by CCl₄. In addition, naringenin increased the liver content of reduced glutathione and the activity of anti‐oxidant enzymes in rats treated with CCl₄. Naringenin attenuated liver inflammation by downregulating CCl₄‐induced activation of tumour necrosis factor (TNF)‐α, inducible nitric oxide synthase (iNOS) and cyclo‐oxygenase (COX‐2) at both the protein and mRNA levels. Naringenin treatment significantly increased NF‐E2‐related factor 2 (Nrf2) and heme oxygenase (HO‐1) expression in injured livers. In rats treated with CCl₄ alone, decreases were seen in nuclear Nrf2 expression and in the mRNA levels of its target genes (e.g. HO‐1, NQO1 and glutathione S‐transferase alpha 3 (GST‐a3)). Together, the results suggest that naringenin can protect the liver against oxidative stress, presumably by activating the nuclear translocation of Nrf2 as well as attenuating the TNF‐α pathway to elicit an anti‐inflammatory response in liver tissue.     

Silymarin attenuates paraquat‐induced lung injury via Nrf2‐mediated pathway in vivo and in vitro

The present study aims to investigate the impacts and mechanisms of silymarin on paraquat (PQ)‐induced lung injury in vivo and in vitro. In in vivo experiments, a total of 32 male Sprague‐Dawley (SD) rats were randomly divided into four groups. The rats were killed on day 3. Histopathological changes in lung tissue were examined using HE and Masson's trichrome staining. Biomarkers of neutrophil activation, pulmonary oedema, pulmonary fibrosis, lung permeability and oxidative stress were detected. Several proinflammatory mediators and antioxidant related proteins were measured. In in vitro experiments, A549 cells were transfected with Nrf2 special siRNA to investigate the roles of Nrf2. The results show that silymarin administration abated PQ‐induced lung histopathologic changes, decreased inflammatory cell infiltration and lung wet weight/dry weight (W/D) ratio, suppressed myeloperoxidase (MPO) activity and nitric oxide (NO)/inducible nitric oxide synthases (iNOS) expression, downregulated hydroxyproline (HYP) levels, reduced total protein concentration and proinflammatory mediator release, and improved oxidative stress (malondialdehyde, MDA; superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GSH‐Px) in lung tissue and serum. Meanwhile, treatment with silymarin upregulated the levels of nuclear factor‐erythroid‐2‐related factor 2 (Nrf2), heme oxygenase‐1 (HO‐1) and NAD(P)H:quinone oxidoreductase‐1(NQO1). However, the addition of Nrf2 siRNA reduced the expression of Nrf2‐mediated antioxidant protein HO‐1 and thus reversed the protective effects of silymarin against oxidative stress and inflammatory response. These results suggest that silymarin may exert protective effects against PQ‐induced lung injury. Its mechanisms were associated with the Nrf2‐mediated pathway. Therefore, silymarin may be a potential therapeutic drug for lung injury.     

Exopolysaccharide of Laetiporus sulphureus var. miniatus downregulates LPS-induced production of NO, PGE2, and TNF-α in BV2 microglia cells via suppression of the NF-κB pathway

Our previous study showed that the exopolysaccharide (EPS) of Laetiporus sulphureus var. miniatus was well characterized and prevented cell damage in streptozotocin-induced apoptosis. However, little is known about the molecular mechanisms underlying its anti-inflammatory effects. Therefore, we attempted in this study to determine whether EPS induces a significant inhibition of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia cells. Our results showed that EPS significantly inhibited LPS-induced pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α), without any significant cytotoxicity. EPS also downregulated mRNA and protein expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-induced BV2 microglia cells. Our data also revealed that EPS treatment significantly reduced translocation of nuclear factor-κB (NF-κB) subunit p65 and its DNA-binding activity in LPS-stimulated BV2 microglia cells. Furthermore, we confirmed by using proteasome inhibitor N-acetyl-l-cysteine (NAC), that the inhibition of NF-κB activity influenced the expression of pro-inflammatory genes in LPS-induced BV2 microglia cells. As expected, NAC suppressed the expression of iNOS, COX-2, and TNF-α by blocking proteasome-mediated degradation. Taken together, our data indicate that EPS inhibits the expression of pro-inflammatory mediators by suppressing NF-κB activity.     

In vivo effects of fenthion on oxidative processes by the modulation of glutathione metabolism in the brain of Oreochromis niloticus

The present study was designed to understand the oxidative stress potential of fenthion, an organophosphate (OP) pesticide and its involvement in glutathione metabolism modulated buthionine sulfoximine (BSO, 50 mg/kg) and N-acetylcysteine (NAC, 100 mg/kg) in the brain of fish, Oreochromis niloticus. A sublethal fenthion concentration (0.45 mg/L) was applied for 24, 48, and 96 h together with injection with BSO or NAC; following treatment, recovery periods for 24, 48, and 96 h were allowed. Total glutathione (tGSH), oxidized glutathione (GSSG), lipid peroxidation, protein level, and GSH-related enzyme activities were analyzed by using spectrophotometric methods. Fenthion in applied concentration did not change GSH levels, but increased GSSG levels. BSO application in fenthion exposure caused a depletion in GSH, while increasing the GSSG levels. Glutathione peroxidase (GPx; EC 1.11.1.9) specific activity increased in fenthion-applied groups at 24-h treatment. gamma-Glutamylcysteinyl synthetase (gamma-GCS; EC 6.3.2.2) was not detected in the brain. NAC injection in fenthion treatment decreased GSH and increased GSSG levels and GST activity. In conclusion, fenthion in sublethal concentration induced an oxidative stress processes in brain. BSO application provided an evidence for the involvement of fenthion in GSH metabolism. NAC elevated the fenthion-induced effects in spite of its antioxidant properties. Recovery period for 96 h was not adequate to eliminate the fenthion-induced changes.     

NSAID-induced small intestinal damage: role of COX inhibition

Indomethacin, the nonselective COX inhibitor, decreased mucosal PGE₂ content and caused damage in the intestine within 24 h, accompanied by increase in intestinal motility, bacterial number and MPO, as well as iNOS activity, together with the up-regulation of COX-2 and iNOS mRNA expression. Neither SC-560 nor rofecoxib alone caused intestinal damage, but their combined administration produced lesions. SC-560, but not rofecoxib, caused intestinal hypermotility, bacterial invasion and COX-2 as well as iNOS mRNA expression, yet the iNOS and MPO activity was increased only when rofecoxib was also administered. Although SC-560 inhibited the PG production, the level of PGE₂ was recovered, in a rofecoxib-dependent manner. Thus, inhibition of COX-1, despite causing intestinal hypermotility, bacterial invasion and iNOS expression, up-regulates the expression of COX-2, and the the COX-2/PGE₂ counteracts deleterious events and maintains the mucosal integrity. This sequence of events explains why intestinal damage occurs when both COX-1 and COX-2 are inhibited.     

(-)-Nyasol (cis-hinokiresinol), a norneolignan from the rhizomes of <Emphasis Type="Italic">Anemarrhena asphodeloides</Emphasis>, is a broad spectrum inhibitor of eicosanoid and nitric oxide production

To assess the anti-inflammatory activity of constituents from the rhizomes of Anemarrhena asphodeloides, (-)-nyasol {cis-hinokiresinol, 4,4-[1Z,3R]-3-ethenyl-1-propene-1,3-diyl]bisphenol} was isolated and its anti-inflammatory activity was examined in lipopolysaccharide (LPS)-treated RAW 264.7 cells and A23187-treated RBL-1 cells. In vivo activity was measured using carrageenan-induced paw edema assay. At > 1 μM, (-)-nyasol significantly inhibited cyclooxygenase-2 (COX-2)-mediated PGE₂ production and inducible nitric oxide synthase (iNOS)-mediated NO production in LPS-treated RAW 264.7 cells, a mouse macrophage-like cell line, but did not affect the expression levels of COX-2 and iNOS. (-)-Nyasol also inhibited 5-lipoxygenase (5-LOX)-mediated leukotriene production in A23187-treated RBL-1 cells. Furthermore, (-)-nyasol potently inhibited carrageenan-induced paw edema in mice (28.6–77.1% inhibition at 24–120 mg/kg). Therefore, (-)-nyasol is a potential new lead compound and may contribute to the anti-inflammatory action of A. asphodeloides, possibly by inhibiting COX-2, iNOS and 5-LOX.     

Ameliorative effects of 3,4-oxo-isopropylidene-shikimic acid on experimental colitis and their mechanisms in rats

The aim of the present study was to investigate the therapeutic effect and mechanism of 3,4-oxo-isopropylidene-shikimic acid (ISA) on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. (50, 100, 200 mg/kg) was administered for 14 days, 1 day after the induction of colitis by TNBS. The colonic injury and inflammation were assessed by macroscopic damage scores and myeloperoxidase (MPO) activity. Malondialdehyde (MDA) and nitric oxide (NO) levels, and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in plasma were measured with biochemical methods. Prostaglandin E₂ (PGE₂) level in colon was determined by radioimmunoassay. Expressions of inducible nitric oxide synthase (iNOS), cyclo-oxygenase-2 (COX-2), inhibitor kappa B-alpha (IκBα) and nuclear factor kappa B (NF-κB) p65 proteins in the colonic tissue were detected with immunohistochemistry. Enhanced colonic mucosal injury, inflammatory response and oxidative stress were observed in the animals clystered with TNBS, which was manifested as the significant increase in colon mucosal damage index, MPO activity, levels of MDA, NO and PGE₂, as well as the expressions of iNOS, COX-2 and NF-κB p65 proteins in the colonic mucosa, and the significant decrease in expressions of IκBα proteins in the colonic mucosa. However, these parameters were found to be significantly ameliorated in rats treated with ISA at given doses, especially at 100 mg/kg and 200 mg/kg. Administration of ISA may have significant therapeutic effects on experimental colitis in rats, probably due to its mechanism of antioxidation, its inhibition of arachidonic acid metabolism and its modulation of the IκBα/NF-κB p65 expression.     

Reactive oxygen species regulate properties of transformation in UROtsa cells exposed to monomethylarsonous acid by modulating MAPK signaling

UROtsa cells exposed to 50 nM monomethylarsonous acid [MMA(III)] for 52 wk (MSC52) achieved hyperproliferation, anchorage independent growth, and enhanced tumorgenicity. MMA(III) has been shown to induce reactive oxygen species (ROS), which can lead to activation of signaling cascades causing stress-related proliferation of cells and even cellular transformation. Previous research established the acute activation of MAPK signaling cascade by ROS produced by MMA(III) as well as chronic up regulation of COX-2 and EGFR in MSC52 cells. To determine if ROS played a role in the chronic pathway perturbations by acting as secondary messengers, activation of Ras was determined in UROtsa cells [exposed to MMA(III) for 0–52 wk] and found to be increased through 52 wk most dramatically after 20 wk of exposure. Ras has been shown to cause an increase in O₂^? and be activated by increases in O₂^?, making ROS important to study in the transformation process. COX-2 upregulation in MSC52 cells was confirmed by real time RT-PCR. By utilizing both antioxidants or specific COX inhibitors, it was shown that COX-2 upregulation was dependent on ROS, specifically, O₂^?. In addition, because previous research established the importance of MAPK activation in phenotypic changes associated with transformation in MSC52 cells, it was hypothesized that ROS play a role in maintaining phenotypic characteristics of the malignant transformation of MSC52 cells. Several studies have demonstrated that cancer cells have lowered superoxide dismutase (MnSOD) activity and protein levels. Increasing levels of MnSOD have been shown to suppress the malignant phenotype of cells. SOD was added to MSC52 cells resulting in slower proliferation rates (doubling time = 42 h vs. 31 h). ROS scavengers of OH also slowed proliferation rates of MSC52 cells. To further substantiate the importance of ROS in these properties of transformation in MSC52 cells, anchorage independent growth was assessed after the addition of antioxidants, both enzymatic and non-enzymatic. Scavengers of OH, and O₂^? blocked the colony formation of MSC52 cells. These data support the role for the involvement of ROS in properties of transformation of UROtsa cells exposed to MMA(III).     

Sodium arsenite induces cyclooxygenase-2 expression in human uroepithelial cells through MAPK pathway activation and reactive oxygen species induction

Arsenic can induce reactive oxygen species (ROS) leading to oxidative stress and carcinogenesis. Bladder is one of the major target organs of arsenic, and cyclooxygenase-2 (COX-2) may play an important role in arsenic-induced bladder cancer. However, the mechanism by which arsenic induces COX-2 in bladder cells remains unclear. This study aimed at investigating arsenic-mediated intracellular redox status and signaling cascades leading to COX-2 induction in human uroepithelial cells (SV-HUC-1). SV-HUC-1 cells were exposed to sodium arsenite and COX-2 expression, mitogen-activated protein kinase (MAPK) phosphorylation, glutathione (GSH) levels, ROS induction and Nrf2 expression were quantified. Our results demonstrate that arsenite (1–10 μM) elevates COX-2 expression, GSH levels, ROS and Nrf2 expression. Arsenite treatment for 24 h stimulates phosphorylation of ERK and p38, but not JNK in SV-HUC-1 cells. Induction of Cox-2 mRNA levels by arsenite was attenuated by inhibitors of ERK, p38 and JNK. Arsenite-induced ROS generation and COX-2 expression were significantly attenuated by treatment with melatonin (a ROS scavenger), but enhanced by DL-buthionine-(S, R)-sulfoximine (BSO, an inhibitor of gamma-glutamylcysteine synthetase (γ-GCS) resulting in lower GSH and increased ROS levels). These data indicate that arsenite promotes an induction of ROS, which results in an induction of COX-2 expression through activation of the MAPK pathway.     

七叶皂苷HK-2细胞毒性的氧化应激机制

目的进一步研究七叶皂苷肾细胞毒性的氧化应激机制及谷胱甘肽(GSH)对七叶皂苷毒性的保护作用。方法以人肾近曲小管上皮细胞(HK-2)为模型,荧光探针法检测七叶皂苷钠(SA)0,10,15,20和25μmol·L-1处理2h后细胞内活性氧(ROS)含量变化;分别用N-乙酰-L-半胱氨酸(NAC)1,5和10mmol·L-1,丁硫氨酸亚砜亚胺(BSO)0.5和2.5mmol·L-1预处理HK-2细胞4h,然后加入SA20μmol·L-1作用24h,DTNB法测定预处理后及加入SA后细胞内GSH的含量;MTT法测定经NAC5mmol·L-1或BSO2.5mmol·L-1预处理及未经预处理的HK-2细胞与SA10,15,20,25,30和40μmol·L-1作用24h后的细胞存活率,并计算IC50值。结果 SA15,20和25μmol·L-1作用2h后,HK-2细胞内ROS含量显著高于正常对照组(P<0.01)。与正常对照组1×106个细胞内的GSH含量(5.1±1.2)nmol相比,BSO2.5mmol·L-1及NAC10mmol·L-1预处理后1×106个细胞内的GSH含量2.8±0.8和(2.7±2.3)nmol均显著降低(P<0.05),其他预处理组GSH含量无显著变化。除NAC10mmol·L-1外,各预处理组与SA20μmol·L-1作用24h后GSH含量显著降低(P<0.01)。与未经预处理的SA20,25和30μmol·L-1细胞存活率〔(83±5)%,(69±5)%和(54±6)%〕相比,经BSO2.5mmol·L-1预处理后,对应的细胞存活率显著降低,分别为〔(69±6)%,(40±13)%和(25±15)%〕(P<0.05),NAC预处理对细胞存活率无显著影响;未经预处理及NAC和BSO预处理的SA的IC50分别为31.3±1.7,23.6±2.7和(34.2±1.5)μmol·L-1。结论七叶皂苷通过氧化应激途径发挥肾细胞毒性,细胞内谷胱甘肽可对其氧化损伤起到保护作用。     

Efficacy of stem cell-based therapies for colistin-induced nephrotoxicity

The increase in infections with multidrug resistant bacteria has forced to return to the use of colistin, antibiotic with known nephrotoxicity. Mesenchymal stem cells (MSCs) are being extensively investigated for their potential in regenerative medicine. This study aimed to investigate the possible protective mechanisms of the MSCs against kidney injury induced by colistin. Forty adult female albino rats were randomly classified into 4 equal groups; the control group, the MSC-treated group (a single dose of 1 ×10⁶ /ml MSCs through the tail vein), the colistin-treated group (36 mg/kg/day colistin was given for 7 days), and the both colistin and MSC group (36 mg/kg/day colistin and 1 ×10⁶ /ml MSCs). Main outcome measures were histopathological alterations, kidney malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and immunohistological autophagy evaluation. MSC repressed the progression of colistin-induced kidney injury as evidenced by the improvement of histopathological alterations and the substantial increase MDA, and decrease SOD and CAT in serum levels. Moreover, MSC resulted in a profound reduction in oxidative stress as manifested by decreased MDA and increased SOD in serum. Notably, MSC suppressed colistin-induced autophagy; it reduced renal levels of Beclin-1, P62 and LC3A/B. Furthermore, MSC decreased renal levels of eNOS. Lastly, MSC efficiently decreased expression of the TUNEL positive cell number. MSC confers protection against colistin-induced kidney injury by alleviating oxidative stress, nitric oxide synthase besides modulating reducing autophagy and apoptosis.     

The crucial protective role of glutathione against tienilic acid hepatotoxicity in rats

To investigate the hepatotoxic potential of tienilic acid in vivo, we administered a single oral dose of tienilic acid to Sprague-Dawley rats and performed general clinicopathological examinations and hepatic gene expression analysis using Affymetrix microarrays. No change in the serum transaminases was noted at up to 1000 mg/kg, although slight elevation of the serum bile acid and bilirubin, and very mild hepatotoxic changes in morphology were observed. In contrast to the marginal clinicopathological changes, marked upregulation of the genes involved in glutathione biosynthesis [glutathione synthetase and glutamate-cysteine ligase (Gcl)], oxidative stress response [heme oxygenase-1 and NAD(P)H dehydrogenase quinone 1] and phase II drug metabolism (glutathione S-transferase and UDP glycosyltransferase 1A6) were noted after 3 or 6 h post-dosing. The hepatic reduced glutathione level decreased at 3-6 h, and then increased at 24 or 48 h, indicating that the upregulation of NF-E2-related factor 2 (Nrf2)-regulated gene and the late increase in hepatic glutathione are protective responses against the oxidative and/or electrophilic stresses caused by tienilic acid. In a subsequent experiment, tienilic acid in combination with l-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of Gcl caused marked elevation of serum alanine aminotransferase (ALT) with extensive centrilobular hepatocyte necrosis, whereas BSO alone showed no hepatotoxicity. The elevation of ALT by this combination was observed at the same dose levels of tienilic acid as the upregulation of the Nrf2-regulated genes by tienilic acid alone. In conclusion, these results suggest that the impairment of glutathione biosynthesis may play a critical role in the development of tienilic acid hepatotoxicity through extensive oxidative and/or electrophilic stresses.     

Arsenic induces mitochondria-dependent apoptosis by reactive oxygen species generation rather than glutathione depletion in Chang human hepatocytes

This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO₂-induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic effect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO₂) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO₂ treatment (0–30 μM) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential (Δψ _m ); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-l-cysteine (NAC, 5 mM) effectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO₂ induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes.     

Biosafety and antioxidant effects of a beverage containing silymarin and arginine. A pilot,human intervention cross-over trial

The study objective was to investigate the potential of a beverage containing silymarin and l-arginine to alter basic physiological and urodynamic parameters in 22 normal healthy men aged 38–59 years. The volunteers drank 500 ml/day beverage without silymarin and l-arginine for 10 days followed, after a 7-day washout period, by the beverage with 400 mg silymarin and 295 mg l-arginine for 10 days. Blood and urine samples were collected on days 0, 10 and 27. The beverages were well-tolerated with no adverse effects. Most of the biochemical, hematological and urodynamic parameters remained unchanged. Total antioxidant capacity, total level of antioxidants, lipoperoxidation products (malondialdehyde), advanced oxidation products of proteins in plasma and glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase levels in erythrocytes were not influenced. Serum γ-glutamyl transferase, malondialdehyde level and activity of glutathione S-transferase in erythrocytes were lowered at day 27 and the concentration of total plasma SH-groups was higher on day 10. Using an ex vivo system, we found that silymarin/silybin at 10–100 μM is able to adsorb onto human erythrocytes and the complexes displayed antioxidant properties as studied using ex situ square-wave voltammetry. The trial showed that silymarin in vivo may protect erythrocytes against oxidative damage.     

R(+)-Methanandamide-Induced Apoptosis of Human Cervical Carcinoma Cells Involves A Cyclooxygenase-2-Dependent Pathway

Purpose  Cannabinoids have received renewed interest due to their antitumorigenic effects. Using human cervical carcinoma cells (HeLa), this study investigates the role of cyclooxygenase-2 (COX-2) in apoptosis elicited by the endocannabinoid analog R(+)-methanandamide (MA). Methods  COX-2 expression was assessed by RT-PCR and Western blotting. PGE₂/PGD₂ levels in cell culture supernatants and DNA fragmentation were measured by ELISA. Results  MA led to an induction of COX-2 expression, PGD₂ and PGE₂ synthesis. Cells were significantly less sensitive to MA-induced apoptosis when COX-2 was suppressed by siRNA or the selective COX-2 inhibitor NS-398. COX-2 expression and apoptosis by MA was also prevented by the ceramide synthase inhibitor fumonisin B₁, but not by antagonists to cannabinoid receptors and TRPV1. In line with the established role of peroxisome proliferator-activated receptor γ (PPARγ) in the proapoptotic action of PGs of the D and J series, inhibition of MA-induced apoptosis was also achieved by siRNA targeting lipocalin-type PGD synthase (L-PGDS) or PPARγ. A role of COX-2 and PPARγ in MA-induced apoptosis was confirmed in another human cervical cancer cell line (C33A) and in human lung carcinoma cells (A549). Conclusion  This study demonstrates COX-2 induction and synthesis of L-PGDS-derived, PPARγ-activating PGs as a possible mechanism of apoptosis by MA. Karin Eichele and Robert Ramer contributed equally to this work.     

Effect of N-acetyl-cysteine, D-penicillamine and buthionine sulfoximine on glutathione levels and CNS oxygen toxicity in rats

The effect of glutathione (GSH) synthesis modulators - L-buthionine sulfoximine (BSO), N-acetyl cysteine (NAC) and D-penicillamine (DPA) - on the susceptibility of rat CNS to O2 toxicity was investigated. The animals were given 5% sucrose or 40 mM solutions of BSO, NAC or DPA in 5% sucrose as drinking water for one week and sacrificed prior to or after exposure to 4.5 ATA O2. The GSH content in brain, liver, lung and blood, and the activity of glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glucose-6-phosphate dehydrogenase (G-6-PD) and superoxide dismutase (SOD) in brain and lungs were measured. The brain GSH content and the enzyme activities were not changed by any of the drugs. BSO decreased the GSH content in all the other tissues; NAC and DPA treatments increased the GSH content in lungs, blood and/or liver. The CNS toxicity threshold as measured by the time of appearance of first electrical discharge (FED) on ECoG recording was not changed by NAC or DPA, but BSO brought about a significant delay in FED time. It is suggested that increased extracerebral GSH levels do not protect against CNS oxygen toxicity, and that BSO provides some protection, probably via a glutathione-independent mechanism.     

Protective effects of seabuckthorn (Hippophae rhamnoides L.) seed oil against carbon tetrachloride-induced hepatotoxicity in mice

The present study examined the protective effects of seabuckthorn (Hippophae rhamnoides L., SBT) seed oil on carbon tetrachloride (CCl₄)-induced hepatic damage in male ICR mice. Our results showed that oral administration of SBT seed oil at doses of 0.26, 1.30, and 2.60 mg/kg for 8 weeks significantly reduced the elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride (TG), and cholesterol at least 13% in serum, and the level of malondialdehyde (MDA) in liver at least 22%, that was induced by CCl₄ (1 mL/kg) in mice. Moreover, the treatment of SBT seed oil was also found to significantly increase the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rd), and GSH content in liver up to 134%. Our study found that the optimal dose of SBT seed oil was 0.26 mg/kg, as the minimum amount exhibiting the greatest hepatoprotective effects on CCl₄-induced liver injury. Overall, the hepatoprotective effect of SBT seed oil at all tested doses was found to be comparable to that of silymarin (200 mg/kg) and have been supported by the evaluation of the liver histopathology in mice.