Agnieszka Szuster-Ciesielska

The inhibitory effect of zinc on cadmium-induced cell apoptosis and reactive oxygen species (ROS) production in cell cultures

The prevention of apoptosis by Zn(2+) is a well-known phenomenon. Both in in vitro and in vivo Zn(2+) supplementation prevents apoptosis induced by a variety of agents, among them by cadmium ions. The target for protective action of Zn ions on cell apoptosis is still unknown. In this paper we have evaluated the effect of in vitro ZnCl(2) supplementation at a concentration corresponding to the physiological level (10 microM) and higher (50 microM), on apoptosis induced with different Cd concentrations in two cell types: HeLa human tumor cell line and bovine aorta endothelial cells (BAECs). We demonstrated that Zn supplementation, especially at 10 microM concentration, significantly inhibited apoptosis in both types of cells. To assess the mechanism involved in the Zn effect we examined the influence of Zn supplementation on Cd accumulation in cells, Cd-induced superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) production. Zn caused 1.2-2.0-fold inhibition of Cd accumulation, 1.2-2.0-fold inhibition of Cd-induced apoptotic cell death, 1.1-2.0-fold decrease in reactive oxygen species (ROS) production in HeLa cells and in BAECs. These results indicate that inhibition of Cd-induced apoptosis in cells by Zn might be due, not only by inhibition of Cd accumulation in cells but, at least in part, to inhibition of Cd-induced production of ROS, which in turn are known as strong inducers of apoptosis.

Serum cytokine levels in alcohol-related liver cirrhosis

Chronic alcoholism complicated by alcoholic liver disease is characterized by activation of the inflammatory response system. To evaluate the role of cytokines in the progress of alcoholic cirrhosis, we assessed serum level of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-8 and the antiinflammatory cytokines IL-2, IL-10, and transforming growth factor (TGF)-beta in patients with compensated and decompensated alcoholic liver cirrhosis. Compensated alcoholic cirrhosis was characterized by increased IL-6 (6.3+/-2.9 vs. HP 2.2+/-1.4 pg/ml in controls) and decreased IL-10 (HP 4.1+/-3.5 vs. 6.4+/-5.4 pg/ml in controls). TNF-alpha, IL-8, and TGF-beta1 levels were comparable to those found in controls. In sera of patients with decompensated alcoholic liver cirrhosis, besides increased IL-6 (11.2+/-7.7 pg/ml), increased concentrations of TNF-alpha (25.1+/-4.5 vs. 9.1+/-7.0 pg/ml in controls) and IL-8 (171.7+/-294.0 vs. 2.7+/-2.9 pg/ml in controls) were also detected. TGF-beta1 and IL-10 levels were similar to those found in controls. These results strongly indicate that a significant derangement of the balance between proinflammatory and antiinflammatory signals is characteristic of compensated and especially of decompensated alcoholic cirrhosis.

Betulin and betulinic acid attenuate ethanol-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS), cytokine (TNF-α, TGF-β) production and by influencing intracellular signaling.

BACKGROUND/AIMS Liver fibrosis has been reported to be inhibited in vivo by oleanolic and ursolic acids. However, the mechanisms of the action of those triterpenoids are poorly understood. In this study, we aimed to determine the antifibrotic potential of other triterpenes, betulin and betulinic acid, and to characterize their influence on the signal transduction pathways involved in ethanol-activated hepatic stellate cells (HSCs). METHODS Investigated was the influence of preincubation of rat HSCs with betulin and betulinic acid, at non-toxic concentrations, on ethanol-induced toxicity, migration, and several markers of HSC activation such as smooth muscle α-actin (α-SMA) and procollagen I expression, release of reactive oxygen species (ROS) and cytokines: tumor necrosis factor-α (TNF-α) and tumor growth factor-β1 (TGF-β1), and production of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). To assess the mechanism of the action of those triterpenes, intracellular signals such as nuclear factor-κB (NFκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol were examined. RESULTS In vitro, betulin, but not betulinic acid, protected HSCs against ethanol toxicity. However, both betulin and betulinic acid inhibited the production of ROS by HSCs treated with ethanol and inhibited their migration as well as ethanol-induced TNF-α, and TGF-β1, production. Betulin and betulinic acid down-regulated ethanol-induced production of TIMP-1 and TIMP-2. Betulin and betulinic acid, also decreased ethanol-induced activity of MMP-2. In ethanol-induced HSCs, betulin inhibited the activation of the p38 MAPK and the JNK transduction pathways, while betulinic acid inhibited the JNK transduction pathway only. They also significantly inhibited phosphorylation of IκB and Smad 3 and attenuated the activation of TGF-β1 and NFκB/IκB transduction signaling. CONCLUSION The results indicated that betulin and betulinic acid inhibited ethanol-induced activation of HSCs on different levels, acting as antioxidants, inhibitors of cytokine production, and inhibitors of TGF-β, and NFκB/IκB transduction signaling. Betulin was also inhibitor of both JNK and p38 MAPK signal transduction, while betulinic acid inhibited only JNK. The remarkable inhibition of several markers of HCS activation makes triterpenes, especially betulin, promising agents for anti-fibrotic combination therapies.

Reactive oxygen species production by blood neutrophils of patients with laryngeal carcinoma and antioxidative enzyme activity in their blood

Squamous cell carcinoma of the head and neck is a devastating illness with a severe impact on affected individuals. Several mechanisms may lead to oxidative stress in tumor-bearing patients, among others chronic inflammation. Inflammatory cells, especially macrophages and neutrophil leukocytes, may produce reactive oxygen species (ROS) which participate in carcinogenesis and tumor-associated immunosuppression. The aim of the study presented in this paper was to compare the production of reactive oxygen species (ROS)—superoxide anion (O2−) and hydrogen peroxide (H2O2)—by neutrophils isolated from the blood of 16 patients with larynx carcinoma and 15 healthy controls. The serum activity of superoxide dismutase and catalase as well as the total peroxidase activity in serum have also been estimated. The production of ROS, especially spontaneous and phorbol 12-myristate 13-acetate (PMA)-induced O2−, was relatively higher in the patients with larynx carcinoma than in the healthy controls and increased parallel with the tumor stage (tumor, node, metastasis—TNM staging). The serum activity of catalase and peroxidase was also highest in the patients with stage T3 and T4 larynx carcinoma. After partial or total laryngectomy, a significant decrease in ROS production and the serum activity of catalase and peroxidase was observed. In contrast, the serum level of superoxide dismutase, which had been low prior to surgery, especially in the patients with advanced tumor stages (T3–T4), increased significantly afterwards. The results indicate the existence of oxidative stress in the blood of patients with larynx carcinoma and its significant decrease after partial or total laryngectomy.

Oxidative stress in blood of patients with alcohol-related pancreatitis

To determine the possible role of oxidative stress in alcoholic pancreatitis, the authors measured the ability of blood neutrophils of 22 patients with acute and 20 patients with chronic alcoholic pancreatitis to produce superoxide anion (O2−) and hydrogen peroxide (H2O2), spontaneously and after in vitro stimulation with phorbol ester and compared it with that of neutrophils isolated from the blood of 16 healthy controls. In addition, they measured serum activities of superoxide dismutase, catalase, and the serum concentration of glutathione peroxidase (GPx). Phorbol ester–induced O2− and H2O2 production in neutrophils of patients with acute and chronic pancreatitis was greater than in controls, but these differences, except of superoxide anion production by neutrophils of patients with chronic pancreatitis, were not statistically significant because of large individual differences. Spontaneous resting production of O2− and H2O2 by neutrophils of patients with chronic pancreatitis was significantly greater than in the controls. Superoxide dismutase and catalase activity was greater in sera of both groups of patients with acute and chronic alcoholic pancreatitis than in controls, but GPx concentration was significantly less in the sera of patients with chronic pancreatitis. Impaired GPx production and increased production of O2− and H2O2 by neutrophils may result in increased lipid peroxidation and could play a role in the pathogenesis of chronic alcoholic pancreatitis.

Zinc supplementation attenuates ethanol- and acetaldehyde-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS) production and by influencing intracellular signaling

BACKGROUND/AIMS Zinc has been reported to prevent and reverse liver fibrosis in vivo; however, the mechanisms of its action are poorly understood. We therefore aimed to determine the antifibrotic potential of zinc. METHODS Assessed was the influence of preincubation of rat HSCs with 30 microM ZnCl2 on ethanol- (in the presence of 4-methyl pyrazole (4-MP)) or acetaldehyde-induced toxicity, apoptosis, migration, expression of smooth muscle alpha-actin (alpha-SMA) and procollagen I, release of reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-alpha), tumor growth factor-beta1 (TGF-beta1), metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases (TIMPs) production. Intracellular signals such as nuclear factor-kappaB (NFkappaB), C-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) induced by ethanol and its metabolite were also assessed. RESULTS 30 microM zinc protected HSCs against ethanol and acetaldehyde toxicity and inhibited their apoptosis. Zinc inhibited the production of ROS by HSCs treated with ethanol and acetaldehyde and inhibited their migration. Zinc also inhibited ethanol- and acetaldehyde-induced TGF-beta1 and TNF-alpha production. Zinc down-regulated ethanol- and acetaldehyde-induced production of TIMP-1 and TIMP-2 and decreased the activity of MMP-2. In ethanol- and acetaldehyde-induced HSCs, zinc inhibited the activation of the p38 MAPK as well as the JNK transduction pathways and phosphorylation of IkappaB and Smad 3. CONCLUSION The results indicated that zinc supplementation inhibited ethanol- and acetaldehyde-induced activation of HSCs on different levels, acting as an antioxidant and inhibitor of MAPK, TGF-beta and NFkappaB/IkappaB transduction signaling. The remarkable inhibition of several markers of HCS activation makes zinc a promising agent for antifibrotic combination therapies.

The influence of cadmium and zinc ions on the interferon and tumor necrosis factor production in bovine aorta endothelial cells

The influence of CdCl(2), used at 1, 10 and 100 microM concentration, and ZnCl(2) at 1, 10 and 100 microM concentration on the production of interferon (IFN) and tumor necrosis factor (TNF) in bovine aorta endothelial cells (BAECs) was examined. BAECs were treated with cadmium ions or zinc ions alone or together with cytokine inducers: Newcastle disease virus (NDV) and lipopolysaccharide (LPS). Cadmium ions at 1 and 10 microM concentration, used alone induced a low, but detectable TNF activity in BAECs, and zinc ions at 1, 10 and 100 microM concentration induced both IFN and TNF activity. In contrast to that, cadmium added to BAECs together with the virus or LPS as cytokine inducers significantly inhibited the production of IFN and TNF. Cadmium effect depended on the concentration used, and 1 and 10 microM CdCl(2) partially, but 100 microM cadmium completely inhibited the production of both cytokines. Zinc ions at 1 and 10 microM concentration, which only slightly inhibited the production of both cytokines, did not reconstitute cadmium-depressed IFN and TNF production. These data indicate that cadmium-induced depression of cytokine production in bovine endothelial cells, in response to viral and bacterial stimuli, cannot be reversed by zinc supplementation.

Role of reactive oxygen species (ROS), metalloproteinase-2 (MMP-2) and interleukin-6 (IL-6) in direct interactions between tumour cell spheroids and endothelial cell monolayer

Metastasis is a multistep process involving a variety of direct cell—cell, cell—matrix and paracrine interactions. In the present study, we examined some consequences of direct interaction between tumour cells and endothelial cells in vitro. When multicellular spheroids of two human tumour cell lines (HeLa and Hep‐2) were transferred onto a human umbilical vein endothelial cell (HUVEC) monolayer, a peri‐spheroidal zone of damaged endothelial cells was observed after 24 h co‐culture. To determine the cause of this damage, the production levels of superoxide anion (O2 −), interleukin‐6 (IL‐6) and metalloproteinase‐2 (MMP‐2) were measured both in co‐culture and in monocultures of the tumour cell spheroids and endothelial cells. Attachment of HeLa and Hep‐2 cellular spheroids to the HUVEC monolayer resulted in 1.6‐fold and 2.1‐fold increases in O2 − release, respectively. Also, the MMP‐2 level was five times greater in the co‐culture than in the tumour spheroid monoculture. The increase of IL‐6 in the co‐culture model, on the other hand, was only slight. However, a 2 h preincubation of endothelial cells with LPS (10 μg/ml) prior to the transfer of spheroids induced a significant increase in the production of this cytokine compared to an appropriate control (an LPS‐activated endothelial cell monolayer). These results strongly suggest that both ROS and MMP‐2 are involved in endothelial cell injury when tumour cells cross the endothelial barrier. Moreover, IL‐6, which participates in the inflammatory response, may also be involved in the extravasation of tumour cells.

The effect of steroidal and non-steroidal anti-inflammatory drugs on the cellular immunity of calves with experimentally-induced local lung inflammation

We examined the effect of a single intravenous dose of flumetasone (SAID) and meloxicam (NSAID) treatment of calves with experimentally-induced localized lung inflammation on immunological and hematological variables such as total protein, gamma globulin, hemoglobin (Hb) concentrations, alkaline phosphatase activity, packed red cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts. The influence of drug treatment on the phagocytic activity of WBC and bronchoalveolar lavage (BAL) cells and their ex vivo ability to produce interferon (IFN) and tumor necrosis factor (TNF) after induction with Newcastle disease virus (NDV), as well as on the development of PHA-induced skin delayed hypersensitivity reaction was also determined. Two days after the treatment of calves with experimentally-induced local lung inflammation with flumetasone (5 mg per calf), we observed a significant increase in WBC count, especially neutrophils, and a decrease in gamma globulin concentration, in the percent of blood phagocytic cells and their random migration. Flumetasone treatment also inhibited the development of skin delayed hypersensitivity reaction. In contrast, the treatment of calves with meloxicam (50 mg per calf) did not influence any hematological parameters or skin reactivity. Both drugs, flumetasone and meloxicam, influenced TNF production in ex vivo cultures of blood and BAL cells, inhibiting excessive TNF production induced by local lung inflammation. Contrary to TNF, the treatment of calves with meloxicam and flumetasone enhanced ex vivo IFN production in blood and BAL cells. Histological examination of lung tissue revealed that in control calves (those not treated with anti-inflammatory drugs) and in calves treated with flumetasone, symptoms of stromo-purulent inflammation of pulmonary tissue developed. However, in calves treated with meloxicam, only interstitial inflammation with a slight thickening of interalveolar septa and infiltration of lymphoid cells was observed. These results suggest that in this model of pneumonia, it is more appropriate to use a single dose of meloxicam, rather than flumetasone, to modulate lung inflammation.

Zinc inhibits ethanol-induced HepG2 cell apoptosis

Alcohol consumption produces a variety of metabolic alterations in liver cells, associated with ethanol oxidation and with nonoxidative metabolism of ethanol, among others apoptosis of hepatocytes. As zinc is known as a potent antioxidant and an inhibitor of cell apoptosis, the aim of this paper was to investigate whether zinc supplementation could inhibit ethanol-induced HepG2 apoptosis, and whether this inhibition was connected with attenuation of oxidative stress and modulation of FasR/FasL system expression. The results indicated that zinc supplementation significantly inhibited ethanol-induced HepG2 cell apoptosis (measured by cytochrome c release from mitochondria and caspase-3 activation) by attenuation of reactive oxygen species (ROS) production, increase in the cellular level of GSH, inhibition of ethanol-induced sFasR and FasL overexpression and caspase-8 activation. These results indicate that zinc can inhibit ethanol-induced hepatocyte apoptosis by several independent mechanisms, among others by an indirect antioxidative effect and probably by inhibition of caspase-8 and caspase-9 activation.