Michał Skrzycki

Glutathione level and glutathione-dependent enzyme activities in blood serum of patients with gastrointestinal tract tumors

OBJECTIVES Glutathione (GSH) and enzymes cooperating with it - glutathione peroxidase (GSHPx), glutathione S-transferase (GST) and glutathione reductase (GSHR) - play crucial role in cell defence against reactive oxygen species (ROS), which are implicated in tumor disease. The aim of this study was to determine if neoplastic diseases of gastrointestinal tract may influence blood GSH level and its dependent enzyme activity. DESIGN AND METHODS Blood serum was obtained before and after surgery from patients with gastric, liver and colorectal cancers, and colorectal cancer liver metastases. Lipid peroxidation and GSH levels, and GSH-dependent enzyme activities were determined by means of spectrophotometric methods. RESULTS Increased level of lipid peroxidation and significant differences in GSH level and GSHPx, GST and GSHR activities were observed in serum taken before and after surgery from patients with gastrointestinal tract tumors compared to those in control serum (from healthy blood donors). CONCLUSIONS Increase of lipid peroxidation and changes in GSH level and related enzyme activities, suggest oxidative stress in serum of patients with gastrointestinal tract tumor causes, which probably arise as a result of enormous production of ROS in the system. These alterations reflect the presence of functional defence mechanism against oxidative stress related firmly to the glutathione metabolism.

Protective effect of N-acetyl-L-cysteine against disulfiram-induced oxidative stress and apoptosis in V79 cells.

This work investigated the effect of N-acetyl-L-cysteine (NAC) on disulfiram (DSF) induced oxidative stress in Chinese hamster fibroblast cells (V79). An increase in oxidative stress induced by DSF was observed up to a 200 μM concentration. It was evidenced by a statistically significant increase of both GSH(t) and GSSG levels, as well as elevated protein carbonyl (PC) content. There was no increase in lipid peroxidation (measured as TBARS). DSF increased CAT activity, but did not change SOD1 and SOD2 activities. Analysis of GSH related enzymes showed that DSF significantly increased GR activity, did not change Se-dependent GPx, but statistically significantly decreased non-Se-dependent GPx activity. DSF showed also pro-apoptotic activity. NAC alone did not produce any significant changes, besides an increase of GSH(t) level, in any of the variables measured. However, pre-treatment of cells with NAC ameliorated DSF-induced changes. NAC pre-treatment restored the viability of DSF-treated cells evaluated by Trypan blue exclusion assay and MTT test, GSSG level, and protein carbonyl content to the control values as well as it reduced pro-apoptotic activity of DSF. The increase of CAT and GR activity was not reversed. Activity of both GPx was significantly increased compared to their values after DSF treatment. In conclusion, oxidative properties are at least partially attributable to the cellular effects of disulfiram and mechanisms induced by NAC pre-treatment may lower or even abolish the observed effects. These observations illustrate the importance of the initial cellular redox state in terms of cell response to disulfiram exposure.

Hymenolepis diminuta: experimental studies on the antioxidant system with short and long term infection periods in the rats.

Many helminths cause long-lasting infections, living for several years in mammalian hosts reflecting a well balanced coexistence between host and parasite. There are many possible explanations as to how they can survive for lengthy periods. One possibility is their antioxidant systems, which can serve as defence mechanisms against host-generated oxygen radicals. Therefore, the aim of this experimental study was to examine the antioxidant system in Hymenolepisdiminuta during short (1.5 months young tapeworms) and long (1.5 years old tapeworms) term infection in the rat small intestine. The strobilae of H. diminuta tapeworms (14 young and three old) were divided into three pieces: the anterior part, containing the genital primordiae in the immature segments; the medial part, containing the early uterus in the mature, hermaphroditic proglottids and the terminal part with the mature gravid uterus in the gravid segments. Supernatants of these fragments were used for determination of markers of oxidative stress: concentration of thiobarbiturate reactive substances (TBARS) and of reduced glutathione (GSH), and the activity of antioxidant enzymes: superoxide dismutase (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (GSHPxs), glutathione transferase (GST) and glutathione reductase (GSHR). The results indicated changes in levels of oxidative stress markers and antioxidant enzyme activity in both the young and old forms of H. diminuta. Relatively high activity of SOD (particularly in the anterior part of young tapeworms) was observed, as was increased activity of total GSHPx and a relatively high concentration of GSH in all parts of the tapeworms. These are caused by exposure to increased amount of ROS, which are produced during the inflammatory state. Due to the high activity of antioxidant enzymes, the anterior section of young and old tapeworms is equipped with a very effective antioxidant system. Old organisms also effectively resist oxidative stress due to reduced levels of lipid peroxidation and the high activity of GST, all of which suggest good adaptation to the hostile environment in the hosts intestine.

Altered expression level of Sigma1 receptor gene in human colorectal cancer

Abstract Nonopioid Sigma1 receptor (Sig1R) influences numerous metabolism functions including regulation of ion channels, reaction on stress and response to growth signals. Due to this influence, Sigma1 receptor ligands show anti-proliferative and cytotoxic action on tumor cells. Additionally its increased level is observed in some types of tumors. Colorectal cancer is one of the most common cancers worldwide and its clinical development is well described. The aim of the study was evaluation of Sigma1 receptor mRNA expression level in human colorectal cancer and colorectal cancer liver metastases at different stages of tumor development. The mRNA was isolated from 30 patients: 18 with colorectal cancer (CRC) and 12 with colorectal cancer liver metastases (CRCLM). The cDNA of Sig1R gene was amplified by polymerase chain reaction using specific primers. The level of Sig1R mRNA expression was determined by measurement of optical density. Sig1R expression level was increased in CRC and CRCLM. The highest level of Sig1R mRNA was observed in UICC stage III. We also showed significant interactions of UICC stage and tumor localization with Sig1R expression level. There were no interactions between UICC stage and age of patients, although we observed significantly decreased level of Sig1R mRNA in older patients. Clinical advancement stage, localization of tumor and age of patients seems to be an important factors influencing Sigma1 receptor expression level. It is probably due to double nature of Sig1R action – in certain conditions it could act pro- or antiapoptotic. This action might depend on Sig1R activity resulting from its expression level.

Modulation of antioxidant defense system by the dithiocarbamate fungicides Maneb and Zineb in Chinese hamster V79 cells and the role of N-acetyl-L-cysteine.

Oxidative stress is one of the major factors leading to Maneb- and Zineb-induced disorders. The aim of this in vitro study was to examine (i) the potency of Maneb and Zineb to induce changes in antioxidant enzyme activities in Chinese hamster fibroblasts V79 cells and (ii) the role of N-acetyl-L-cysteine (NAC) in the preventing their action. Maneb increased mitochondrial superoxide dismutase (SOD2) activity but failed to affect the activity of cytoplasmic superoxide dismutase (SOD1), whereas Zineb did not change the activity of any of superoxide dismutases. The activity of catalase (CAT) was reduced only by Zineb. The activity of both glutathione peroxidases (non-Se-GPx, Se-GPx) and glutathione reductase (GR) was decreased after exposure to these agents. After NAC pre-treatment Maneb increased the activity of GR, whereas the activity of non-Se-GPx was decreased as compared to that in NAC-treated cells. On the other hand, the activity of both SODs and CAT was decreased. Zineb decreased the activity of both GPxs and SOD2 with a concomitant increase in CAT activity comparing to NAC-treated cells. The results obtained thus suggest that Zineb acts by another mechanism, than Maneb does, and that one of the mechanisms of NAC protection against Maneb or Zineb-induced effects in V79 cells is its impact on enzymatic defense. Activity of GR, SOD2, and GPxs are the most affected enzymes.

Superoxide dismutase mRNA and protein level in human colorectal cancer

Impairments of antioxidant enzyme expression are often concomitant with the onset of cancer. Due to epigenetic factors causing an inflammatory state the gastrointestinal tract can become exposed to reactive oxygen species. The purpose of our work was to evaluate mRNA and protein levels of superoxide dismutase isoenzymes in human colorectal adenocarcinoma due to its clinical advancement, and in colorectal cancer liver metastases. Evaluation of SOD expression in regard to CRC advancement, seems useful for clinical applications due to different tumor cells sensitivity to reactive oxygen species based treatment. Studies were conducted on a group of 27 patients: 15 diagnosed with colorectal adenocarcinoma and 12 diagnosed with colorectal cancer liver metastases. The mRNA level was determined by RT-PCR, and protein level by Western blotting. We observed significant (P≤0.05) changes of mRNA and protein level of SOD isoenzymes in subsequent stages of colorectal adenocarcinoma advancement and in colorectal cancer liver metastases. Differences in mRNA and protein level of SOD isoenzymes in colorectal adenocarcinoma and its liver metastases indicates that SOD participate in adaptation of tumor cells to oxidative stress, and maintain certain level of ROS, necessary for appropriate cell proliferation. Expression of superoxide dismutase isoenzymes seems to be regulated not only at transcriptional level, but also posttranscriptional.

Multiple protective functions of sigma1 receptor.

The Sigma Receptor 1 (sig-1R) is a protein present in numerous normal tissues, such as brain, retina, lens, liver, lung, heart, but also in many tumor lines. Its amino acid sequence is homologous to fungal C-8,7 sterol isomerase, but it has no known homology with mammalian proteins and does not possess sterol isomerase activity. It is localized in plasma and ER membranes, and its exact function is not clarified as of yet. Last reports point to its participation in regulation of ionic channels activity, particularly calcium channels. Application of numerous synthetic ligands of sigma1 receptor provided means to study its protective effects and metabolic functions in different tissues. This review describes influence of sigma1 receptor on various aspects of cellular metabolism, such as calcium signalling, mitochondrial functions, oxidative stress, survival and apoptotic pathways, and tumor cells proliferation.

Enzymatic antioxidant system in the cestode Hymenolepis diminuta after chronic infection of the rat

BackgroundThe aim of the present paper was to describe the enzymatic antioxidant system in Hymenolepis diminuta collected from rats exposed to chronic cestode invasion.MethodologyWe dissected different tissues of H. diminuta (immature proglottids, genital primordia, hermaphroditic proglottids, early uterus, and gravid uterus) and studied activity of: superoxide dismutases (SOD1 and SOD2), catalase (CAT), glutathione peroxidases (non-Se-dependent GSHPx and Se-dependent GSHPx), glutathione-S-transferase (GST) and glutathione reductase (GSHR), and oxidative stress markers — reduced glutathione (GSH), and the lipid peroxidation level (TBARS).ResultsWe demonstrated changes in antioxidant enzyme activities and levels of oxidative stress markers in different tissues of the parasite. The levels of TBARS and GSH indicate that oxidative stress occurred in tissues located proximal to the intestine wall. Activity of SOD1 was high in all parts of H. diminuta, but the GST activity was the highest of all studied antioxidant enzymes. SOD2 activity differed significantly in various parts of H. diminuta. Significant differences were observed for nonSeGSHPx and activity of other GSH-dependent enzymes was generally similar in all the tissues.ConclusionsOur results show that the enzymatic antioxidant system of H. diminuta, allows the parasite to adapt and live under conditions of chronic oxidative stress. It suggests an oxidative-antioxidative balance during interactions between parasite and host.

Effect of different concentrations of oxygen on expression of sigma 1 receptor and superoxide dismutases in human colon adenocarcinoma cell lines

Abstract Context: Tumor cells due to distance from capillary vessels exist in different oxygenation conditions (anoxia, hypoxia, normoxia). Changes in cell oxygenation lead to reactive oxygen species production and oxidative stress. Sigma 1 receptor (Sig1R) is postulated to be stress responding agent and superoxide dismutases (SOD1 and SOD2) are key antioxidant enzymes. It is possible that they participate in tumor cells adaptation to different concentrations of oxygen. Objective: Evaluation of Sig1R, SOD1, and SOD2 expression in different concentrations of oxygen (1%, 10%, 21%) in colon adenocarcinoma cell lines. Materials and methods: SW480 (primary adenocarcinoma) and SW620 (metastatic) cell lines were cultured in standard conditions in Dulbecco’s modified Eagle’s medium for 5 days, and next cultured in Hypoxic Chamber in 1% O2, 10% O2, 21% O2. Number of living cells was determined by trypan blue assay. Level of mRNA for Sig1R, SOD1, and SOD2 was determined by standard PCR method. Statistical analysis was conducted using Statistica 10.1 software. Results: We observed significant changes in expression of Sig1R, SOD1, SOD2 due to different oxygen concentrations. ANOVA analysis revealed significant interactions between studied parameters mainly in hypoxia conditions in SW480 cells and between Sig1R and SOD2 in SW620 cells. It also showed that changes in expression of studied proteins depend significantly on type of the cell line. Conclusion: Changes of Sig1R and SOD2 expression point to mitochondria as main organelle responsible for survival of tumor cells exposed to hypoxia or oxidative stress. Studied proteins are involved in intracellular response to stress related with different concentrations of oxygen.

Changes of enzymatic antioxidant system in the small intestine of rats after the chronic invasion by Hymenolepis diminuta (Cestoda, Hymenolepididae)

SummaryThe aim of this study was to evaluate changes in the enzymatic antioxidant system in rat small intestine caused by invasion of tapeworms Hymenolepis diminuta. The study material consisted of samples of the rats small intestine after short- (1.5-months) and long-term (1.5-years) larvae invasion of tapeworm H. diminuta. In tissue extracts the concentration of oxidative stress markers (GSH and TBARS) and activity of antioxidant enzymes (SOD, CAT, total GSHPx, SeGSHPx, GST and GSHR) were determined. Changes demonstrated for GSH and TBARS level and the activity of antioxidant enzymes in the small intestine in rats indicate the induction of oxidative stress and weakening of antioxidant defense mechanisms, after both short- and long-term invasion of H. diminuta tapeworms. Observed profile of antioxidant enzymes activity in the small intestine of rats after prolonged exposure to direct or indirect contact with H. diminuta tapeworms points to the adaptation of the definitive host to oxidative stress and defense against parasitic invasions.