Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed.

Some lung cellular parameters reflecting inflammation after combined inhalation of amosite dust with cigarette smoke by rats.

Cellular changes were followed in lung cell suspensions after 175 day inhalation by rats of concentrations 30 mg/m3 or 60 mg/m3 of amosite asbestos every second day combined with daily exposure to cigarette smoke at 30 mg of total particulate matter (TPM)/m3 air. Concomitantly, lung inflammation was assessed by changes in the bronchoalveolar lavage fluid (BALF). A dose-dependent rise in the BALF inflammatory parameters was found. The rise of the proportion of binucleate (BNC) and multinucleate cells (MNC) in lung cell suspensions was also dose-dependent. It is concluded that, in the experimental assessment of effects of fibrogenic dusts, the number of BNC and of MNC in lung cell suspensions may serve as a useful semiquantitative biomarker of the inflammation.

[Relation between fatty acid composition, vitamin e and malondialdehyde levels, and activity of antioxidant enzymes in the blood]. / Vzájomný vztah zlozenia mastných kyselín ku koncentrácii vitamínu E, k hladine malóndialdehydu a k aktivitám antioxidacných enzýmov v krvi.

The aim of our study was to determine the composition of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in erythrocytes and to investigate their relation to the concentration of vitamin E (Vit E) in the plasma, level of malondialdehyde (MDA) in the serum and activities of antioxidant enzymes (AE) in erythrocytes. We examined 128 healthy volunteers (57 men and 71 women) at the ages ranging between 20 and 72. Fatty acids were assessed by gas chromatography, vitamin E by the HPLC method, MDA by the spectrophotometric method, GSH-Px by the kinetic method, CU, Zn-SOD by the Randox test. We have detected the following order in the proportion of fatty acids: SFA > PUFA > MUFA. The increase in PUFA and MUFA was in direct correlation with the concentration of MDA and AE activity, however in reverse correlation with Vit E. A reverse correlation was observed in the increase in SFA. We have found an increase in lipoperoxidation due to the increase in PUFA in erythrocytes in the group of healthy people. This state is concommited by an increase in the activity of antioxidant protection for the maintenance of the balance state. The authors assume that a decrease in Vit E can be caused by its increased "consumption" as the most effective antioxidant in lipid environment. (Tab. 2, Fig. 1, Ref. 27.)

[Relation between levels of vitamins C, E, A and beta-carotene and activity of antioxidant enzymes in the blood]. / Vztah hladín vitamínov C, E, A a beta-karoténu k aktivite antioxidacných enzýmov v krvi.

UNLABELLED: Antioxidant protection is procured by many enzymatic and non-enzymatic factors which maintain the physiological level of reactive forms of oxygen. The aim of this study was to determine the levels and relationship between concentrations of the selected non-enzymatic antioxidants in healthy people (vitamin C--Vit C, vitamin E--Vit E, beta-carotene-beta-Car and vitamin A--Vit A) in the serum, antioxidant enzymes, CuZn-superoxide dismutase--SOD, catalase CAT, glutathione peroxidase--GPx) in erythrocytes and malondialdehyde (MDA) in the serum. We have examined 128 volunteers (57 men and 71 women) at the age ranging between 20 and 72. The average levels of vitamins and beta-Carotene were detected near the lower level of normal values or below it, whilst the highest interindividual differences were detected in Vit C and Vit E. The level of Vit E indirectly correlated with SOD, GPx, MDA, but directly with CAT. On the contrary, the levels of Vit A directly correlated with SOD, GPx, MDA and indirectly with CAT. The levels of Vit C and beta-Carotene had no relation with the activities of antioxidant enzymes and the level of MDA. The direct correlation was between the levels of Vit C and Vit E, and Vit C and beta-Carotene. CONCLUSIONS: 1. A decrease in average levels of Vit A, C, E and beta-Carotene in the blood of the investigated group of healthy persons indicates their lower antioxidant protection. 2. The relation between Vit E, MDA, activities of GPx and SOD indicates the presumed mutually supplementing effect of these antioxidants in liquidation of products of lipoperoxidations. (Tab. 2, Fig. 3, Ref. 17.)

[Levels of Cu, Zn, Se and their relation to levels of ceruloplasmin and the activity of antioxidative enzymes]. / Koncentrácia Cu, Zn, Se a ich vztah k hladine ceruloplazmínu a aktivite antioxidacných enzýmov.

BACKGROUND: Essential trace elements copper, zinc and selenium are important parts of antioxidant enzymes as superoxide dismutase, glutathione peroxidase as well as of transport protein with antioxidant properties-ceruloplasmin. Mentioned trace elements may affect antioxidant defence system. AIM OF STUDY: The aim of our study was to estimate serum levels of copper, zinc, and selenium in a group of healthy subjects and to correlate them with erythrocyte activity of Cu,Zn-SOD, GSH-Px and serum levels of ceruloplasmin. METHODS: We examined a group of 128 healthy volunteers (57 men and 71 women) aged 20 to 74 years. The analyses of trace elements we performed by atomic absorption spectrophotometry, Cu,Zn-SOD were detected by Randox test, GSH-Px by kinetic method and ceruloplasmin was measured immunoelectrophoretically. RESULTS: We have not found any correlations between investigated trace elements, age and sex. Average serum levels were as follows: copper (17.26 +/- 2.71 mumol/L), zinc (15.30 +/- 1.92 mumol/L), and selenium (0.79 +/- 0.19 mumol/L). There was a weak linear correlation between serum levels of copper and ceruloplasmin. CONCLUSION: There was no relationship between serum copper and zinc levels and Cu,Zn-SOD activity, and serum concentration of selenium and GSH-Px activity in the group of healthy subjects. A moderate linear correlation was estimated between serum values of copper and ceruloplasmin. (Tab. 2, Fig. 4, Ref. 29.)

[Ulcerative colitis: activity of antioxidant enzymes of the colonic mucosa]. / Colite ulcéreuse: activité des enzymes antioxydantes de la muqueuse colique.

OBJECTIVES: Reactive oxygen species are regarded as mediators of inflammation in ulcerative colitis. Antioxidant enzymes are the primary defense against reactive oxygen species. We investigated the activities of antioxidant enzymes-superoxide dismutase, catalase and glutathion peroxidase-in colonic mucosa biopsy specimens obtained at different phases of the disease in patients given vitamin E therapy or not and in controls. PATIENTS AND METHODS: Biopsy specimens were obtained from 29 patients with ulcerative colitis and from 18 healthy controls. Ten patients on maintenance drug therapy were also given a 300 mg daily oral dose of vitamin E. RESULTS: Enzyme activities were significantly increased in inflamed non-polypous colonic mucosa in patients with active ulcerative colitis, while they were similar to activities observed in controls during quiescent phases of the disease. Antioxidant enzyme activities in specimens from inflammatory polyps remained high even in patients in clinical remission. Administration of vitamin E resulted in a significant reduction of glutathion peroxidase activity in the colonic mucosa. CONCLUSION: These findings indirectly confirm that important quantities of reactive oxygen species are formed in ulcerative colitis. Further studies are needed to elucidate the relationship of these changes with the pathogenesis of ulcerative colitis.

[Adenomatous and inflammatory colorectal polyps: antioxidative enzyme activity in the colon]. / Adenomatózne a zápalové polypy kolorekta: aktivita antioxidacných enzýmov v kolone.

BACKGROUND: Reactive types of oxygen play an important part also in carcinogenesis. Antioxidant enzymes are the primary defence against their damage. The objective of the present work was to glutathione peroxidase in the mucosa and polyps of the colon in subjects with colorectal adenoma and idiopathic proctocolitis. METHODS AND RESULTS: The authors examined 18 controls, 43 patients with colorectal adenoma and 12 subjects with idiopathic proctocolitis. During endoscopy bioptic specimens of the mucosa and from polyps were taken for histological and enzymological examination: Cu/Zn superoxide dismutase, catalase, glutathione peroxidase. In subjects with colorectal adenoma a raised glutathione peroxidase activity was found in the colon and an elevated activity of superoxide dismutase in the adenoma. In patients with idiopathic proctocolitis in the stage of clinical remission in the mucosa a lower glutathione peroxidase activity was found but a high activity of all enzymes was recorded in the inflamed polyps. CONCLUSIONS: The cause of elevated activities of antioxidant enzymes in subjects with colorectal adenoma in the colonic mucosa and in adenomas is not known and calls for further studies. In patients with idiopathic proctocolitis the increased level of antioxidant enzymes in the mucosa is probably produced by a higher production of reactive oxygen types by activated leucocytes in the inflamed tissues.

[Levels of erythrocyte glutathione and ceruloplasmin and transferrin in the serum and their role in antioxidant protection]. / Koncentrácia glutatiónu v erytrocytoch, ceruloplazmínu a transferínu v sére a ich úloha pri antioxidacnej obrane.

All organisms are protected from harmful reactive oxygen which is produced also under physiological conditions by a complex antioxidative system. Our work was aimed at the ascertainment of the level of reduced and oxidated glutathione in erythrocytes of healthy people, the concentrations of ceruloplasmin (GSH) and transferrin (GSSG) in the serum, as well as the investigation of the relationship to antioxidative enzymes ---Cu, Zn-superoxide dismutase (SOD), catalase (CAT) and Se-glutathione-peroxidase (GPx) in erythrocytes. We discovered a mutual direct linear correlation between the levels of GSH, GSSG, CPL and TRF, indirect linear relation between the concentrations of TRF, GSH, GSSG and activities of SOD and GPx, between the concentrations of CPL and GPx activities, and a direct linear relation between concentrations of GSH and TRF with CAT activity. The results indicate to a mutual dependence of investigated nonenzymatic antioxidative factors and antioxidative enzymes. (Tab2, Fig. 4, Ref.13.).

[Antioxidative enzyme activity in the blood of healthy persons]. / Aktivita antioxidacných enzýmov v krvi zdrav ch osôb.

BACKGROUND: Under physiological conditions the dynamic balance between the reactive oxygen production and detoxication is maintained. OBJECTIVES: The study was aimed at the detection of the activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) in erythrocytes and the level of malondialdehyde (MDA) in the serum of healthy subjects, their relation to age, body weight, sex, smoking, and the possible correlation between individual parameters. METHODS: The authors have examined 128 healthy volunteers (57 men and 71 women) from 20 to 72 years of age. The results were statistically evaluated by means of the ANOVA programme. RESULTS: The group of healthy people had a large dispersion of interindividual activities of antioxidant enzymes and MDA levels. Age and weight were reversely correlated to the CAT activity in erythrocytes. The CAT activity was significantly higher in women than in men, and the GPx activity was lower in smokers than in non-smokers. The rest of investigated parameters were not significantly influenced by sex of examined people and smoking. Mutual correlation between individual enzymes were highly significant. CONCLUSIONS: A large dispersion of activities of the investigated enzymes and MDA levels in healthy individuals do not allow to make a standard use of these parameters in clinical practice. (Tab. 5, Fig. 2, Ref. 20.).

Increased antioxidant enzyme activities in the colorectal adenoma and carcinoma.

Most colon carcinomas are preceded by an adenomatous polyp--adenoma-carcinoma sequence. Active oxygen species (AOS) can play a role in the pathogenesis of this process. Antioxidant enzymes (AE) are the primary defense against the deleterious effect of AOS. Activities of AE in 56 individuals with colorectal adenoma (CA), 29 individuals with colorectal carcinoma (CC) and in 24 control subjects were examined. Biopsy specimens from the non-neoplastic colonic mucosa and from the CA and CC were taken during colonoscopy for histological and enzymological analysis. Activities of following AE were estimated: CuZn-superoxide dismutase (CuZn-SOD), catalase (CAT) and glutathione peroxidase (GPx). It was found that individuals with CA and CC were characterized by: (1) increased activities of CAT and GPx in non-neoplastic mucosa, that persisted in some of the patients even after removal of tumors; (2) increased activities of CuZn-SOD, CAT and PGx in CA and CC tissues. It can be inferred that the accumulation of peroxides in the non-neoplastic colonic mucosa induced higher activities of CAT and GPx. The reasons of high activities of all AE in the tissues of CA and CC and their relation to carcinogenesis are not clear and require further studies.

Effect of intake of exogenous vitamins C, E and beta-carotene on the antioxidative status in kidneys of rats with streptozotocin-induced diabetes.

We studied the effect of supplementation with vitamins C, E and beta-carotene (PARABION, produced by Syndipharma) on antioxidative status in kidneys of male Wistar rats with diabetes induced by intravenous application of streptozotocin (45 mg.kg-1 of body weight). The animals received subtherapeutic doses of Insulin Interdep (6 U.kg-1 of body weight). A significant decrease of malondialdehyde (MDA), reduced (GSH) and oxidized (GSSG) glutathione and reduction of the activities of Se-glutathione peroxidase (Se-GSH-PX, EC. 1.11.1.9.) and glutathione S-transferase (GST, EC. 2.5.1.18.) were observed in kidneys of diabetic rats treated with these vitamins. On the contrary, the activity of CuZn-superoxide dismutase (CuZn-SOD, EC. 1.15.1.1) and the level of vitamin C (vit. C) increased significantly. No changes were observed for vitamin E (vit. E), beta-carotene and catalase (CAT, EC. 1.11.1.6). Supplementation with vitamins C, E and beta-carotene resulted in an improvement of antioxidative status of kidneys of rats with streptozotocin-induced diabetes.

[Activity of the antioxidant system in patients with idiopathic proctocolitis and the effect of 5-aminosalicylic acid (Salofalk)]. / Aktivita antioxidacného systému pacientov s idiopatickou proktokolitídou a vplyv kyseliny 5-aminosalicylovej (Salofalk).

There is a close relationship between inflammation and reactive oxygen species (ROS). Primary source of ROS are activated leucocytes. Antioxidant system protects organism against the deleterious effect of ROS. The aim of the present study was to follow the activity of antioxidant system in blood and colonic mucosa of 17 patients with idiopathic proctocolitis. All patients were treated with 5-aminosalicylic acid (Salofalk), 7 patients with a combination of prednison. The following biochemical parameters were ascertained: malondialdehyde (MDA) and ceruloplasmin in serum, glutathione (GSH) and activities of superoxide dismutase, catalase and glutathione peroxidase in erythrocytes and colonic mucosa. Before treatment there was found: an increase of MDA and all antioxidant enzyme activities, an a decrease of GSH. After 3 weeks of therapy in 59% of patients, initial clinical remission was observed without serious improvement of biochemical parameters. After 10 weeks of therapy the clinical course improved in all patients, a significant decrease of MDA and activities of all antioxidant enzymes as well as an increase of GSH were stated. The authors assume that IP was positively affected by 5-ASA, and in some patients by its combination with prednisom. The effect of 5-ASA is based on its known antiinflammatory impact and its functioning as a "scavanger" of free radicals (or ROS). (Tab. 1, Fig. 3, Ref. 23.)

[Antioxidant enzyme activity in the gastric mucosa in precancerous conditions]. / Aktivita antioxidacných enzýmov v sliznici zalúdka u prekanceróznych stavov.

BACKGROUND: The primary defense against oxidation damage of tissues are anti-oxidant enzymes, e.g. superoxide dismutase, catalase and glutathione peroxidase. Some non-enzymatic substances have a significant anti-oxidant action (e.g. vitamin C, E, beta-carotene and others). The objective of the present work was to follow up the Cu/Zn superoxide dismutase activity, catalase and glutathione peroxidase (anti-oxidant enzymes of the gastric mucosa) in subjects with the risk of developing gastric cancer, e.g. those suffering from atrophic gastritis, hyperplastic polyps and gastric adenoma. METHODS AND RESULTS: The authors examined 80 subjects (50 men and 30 women) aged 25 - 71 years. In all during endoscopic examination bioptic specimens of the mucosa were taken at standard sites of the gastric corpus and antrum for histological and enzymological examination. Enzymological examination: activity of Cu/Zn-superoxide dismutase (Randox Lab. Ltd. GB kit), catalase activity (modified method of Cavarocchia et al.) and glutathione peroxidase activity (method according to Paglia and Valentine). The Cu/Zn-superoxide dismutase activity was elevated in the group of patients with gastritis after gastrectomy (67%) and with gastric adenoma (35%), the catalase activity in patients with gastritis after gastrectomy (40%) and the glutathione peroxidase activity in patients with the diagnosis of gastritis after gastrectomy (185%), atrophic gastritis (46%) hyperplastic polyp (50%) and gastric adenoma (50%). CONCLUSIONS: The increased activity of anti-oxidant enzymes was due to a higher concentration of the superoxide anion radical, hydrogen peroxide and organic peroxides (lipoperoxides); the source of active types of oxygen are phagocytic leucocytes in the chronically inflamed gastric mucosa.

The activity of Cu/Zn-superoxide dismutase and catalase of gastric mucosa in chronic gastritis, and the effect of alpha-tocopherol.

Activity of Cu/Zn superoxide dismutase (SOD) and catalase (CAT) in gastric mucosa was examined in 12 control subjects and 48 patients with various degree of chronic gastritis. Bioptic specimens of mucosa for enzymologic assays were sampled during gastroscopic examination after overnight fasting. Compared to the control group, a significant increase of SOD activity was observed in samples from superficial gastritis and only insignificant changes in samples from atrophic gastritis, while a remarkable and significant increase in the activity of both SOD and CAT was observed in samples from gastritis after partial gastrectomy. Application of vitamin E significantly reduced activities of SOD and CAT in subjects with chronic gastritis. The authors suppose that the increase in the activities of antioxidant enzymes was caused by an increased formation of superoxide anion radical and hydrogen peroxide, both originating from phagocyting leucocytes in inflamed mucosa. The reduced activity of SOD and CAT after application of vitamin E indicates an inhibition of the formation of active oxygen species in gastric mucosa. Relation of chronic gastritis and elevated concentrations of active oxygen species to gastric carcinogenesis is discussed in the report. (Tab. 2, Fig. 1, Ref. 22.)

Increased mucosal antioxidant enzyme activities in chronic gastritis and benign gastric polyps.

Gastroscopy with gastric biopsy was performed in 109 individuals aged 25-71 years. Activities of three antioxidant enzymes were assayed in biopsy specimens: Cu/Zn superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Patients were classified according to the endoscopic and histological findings in the following groups: normal findings (N), superficial gastritis (SG), mild (MAG) and severe (SAG) atrophic gastritis, gastritis after partial gastrectomy (PGG), hyperplastic polyp (HP), and gastric adenoma (A). Compared with the N group, increased activity of SOD was found in groups SG (+37%), PGG (+67%) and A (+35%), increased CAT activity in PGG (+40%), and increased GSH-Px activity in groups SG (+57%), SAG (+46%), PGG (+185%), HP (+50%) and A (+50%). Increased activity of antioxidant enzymes could be induced by higher concentrations of superoxide anion radicals, hydrogen peroxide and lipid peroxides, produced by phagocytic leucocytes or by polyunsaturated fatty acid in cellular membranes of gastric mucosa. The relation of reactive oxygen species to the induction of precancerous conditions and to carcinogenesis of the stomach requires further study.

Gastric mucosal antioxidant activity in patients at increased risk of gastric cancer.

In 68 subjects the activities of Cu/Zn-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were investigated in gastric mucosa. The patients were classified according to the histological finding into following groups: 12 with normal finding (N), 16 with superficial gastritis (SG), 13 with mild atrophic gastritis (MAG), 19 with severe atrophic gastritis (SAG) and 8 with gastritis after partial gastrectomy (PGG). The comparison of groups SG, MAG, SAG and PGG with the group N revealed the following changes: in SG increased SOD and GSH-Px, in MAG and SAG no significant changes, and in PGG increase in SOD, CAT and GSH-Px were observed. It was supposed that increased enzymatic activities were caused by higher concentration of active oxygen species produced by phagocytizing leukocytes in inflamed gastric mucosa. Administration of vitamin E resulted in significant reduction of SOD and CAT activities, on the other hand GSH-Px activity significantly increased. The explanation of this effect of vitamin E requires further studies. A prolonged interaction of active oxygen species with chemical carcinogens (N-nitroso- or diazonium compounds, PAH) can exhibit a significant promoting effect on the development of intestinal type of gastric cancer from its precancerous conditions, above all after partial gastrectomy.