Biological durability and oxidative potential of man-made vitreous fibres as compared to crocidolite asbestos fibres

In this study we investigated relationships between redox properties and biodurability of crocidolite asbestos fibres and three different man-made vitreous fibres (MMVF): traditional stone wool fibres (MMVF 21), glass fibres (MMVF 11) and refractory ceramic fibres (RCF). Each fibre type was incubated up to 22 weeks in four different incubation media: gamble solution (GS) pH 5.0 and pH 7.4, representing blood plasma without proteins, and surfactant-like solution (SLS) pH 5.0 and pH 7.4. During incubation time aliquots of incubation mixtures were removed and analysed in a biochemical model reaction, mimicking activated phagocytes. In addition, changes of fibre morphology and chemical composition were examined using SEM- and EDX-technology. In the presence of crocidolite asbestos fibres and MMVF 21 the formation of OH*-radicals according to the Haber-Weiss sequence could be demonstrated, whereas MMVF 11 and RCF showed no reactivity. Crocidolite asbestos fibres exhibited a significant higher activity compared with the stone wool fibres at the onset of incubation. The oxidative capacities of these fibre types were shown to depend on both specific surface area and iron content. The oxidative potentials of crocidolite asbestos fibres as well as MMVF 21 were not constant during incubation over several weeks in each incubation medium. The reactivities showed sinoidal curves including reactivities much higher than those at the onset of incubation time. These irregular changes of oxidative capacity may be explained by changes of the redox state of fibre surface-complexed iron. Furthermore our results showed clear differences between incubation of fibres in GS and SLS, respectively, indicating that phospholipids play an important part in fibre dissolution behaviour and oxidative reactivity. In conclusion we suggest, that biodurability testing procedures should not exclusively concentrate on dissolution rates of fibres. They should include fibre characteristics concerning known pathogenic mechanisms to evaluate the real toxic potential of the fibre type looking at. Secondly we suggest, that phospholipids should be constituents of incubation liquids used for standardised fibre biodurability test procedures thus representing more realistic incubation conditions.     

Protein-nitrogen metabolism in the liver of rats intraperitoneally administered carminomycin]

It was found in the experiments with rats that in response to carminomycin administration in a single LD50 and the therapeutic doses during the treatment course the intracellular fond of the amino acids in the liver tissue increased, the autolytic processes activated, the activity of the transamination enzymes, histidine and ammonia lyases increased. The level of the residual nitrogen increased mainly at the expense of increased ammonia production and urea levels. These changes were more pronounced when the antibiotic was administered in LD50. Independent of the administration multiplicity and the antibiotic dosage they were of reversible nature and mainly disappeared by the end of the observation.     

Effect of orally and intraperitoneally administered plant lectins on food consumption of rats

A panel of orally administered lectins (100 mg/kg b.w.) of different binding specificities was tested for suppression of voluntary food consumption in prefasted rats. PHA isolectins (Phaseolus vulgaris) and RPA-I (Robinia pseudoacacia) were found to exert a marked and significant effect, but two other gut-binding lectins, i.e. SBA (Glycine max) and WGA (Triticum vulgar) and several non-binding lectins were ineffective. In cannulated rats PHA infused into the duodenum induced food suppression, i.e. binding of the lectin to the mouth or stomach was unnecessary. Suppression of food consumption lasted through the whole nocturnal feeding period, control (BSA) and experimental (PHA) curves of cumulative food consumption showed a V-like divergence. Suppression by PHA or RPA-I showed very similar time courses, but a long-lasting inhibition of gastric emptying was only observed in the RPA-treated animals. Intraperitoneally administered lectins suppressed food consumption much more effectively than the oral ones, whereas Galanthus nivalis agglutinin (ONA) had little or no effect. It is concluded that lectins can be used as effective tools for the modulation of food consumption and gastric emptying in experimental animals.     

Protective role of intraperitoneally administered vitamin E and selenium in rats anesthetized with enflurane

The aim of this investigation was to determine levels of liver vitamins A and E and blood biochemical and hematological parameters in the enflurane anesthesia of rats. Fifty adult male Wistar rats were used in this study. All rats were randomly divided into five groups. The first and second groups were used as the control and anesthesia control groups, respectively, and only the placebo was intraperitoneally injected. The third group was intraperitoneally administered with vitamin E (dl/-α-tocopheryl acetate, 100 mg/kg body weight), the fourth group with Se (Na₂SeO₃ 1.5 mg/kg body weight), and the fifth group with vitamin E and Se (dl-α-tocopheryl acetate, 100 mg/kg body weight + Na₂SeO₃ 1.5 mg/kg body weight). This administration was done for three times with overday intervals and the second, third, forth, and fifth group rats were taken to enflurane anesthetise for 2 h. The liver vitamin E level was slightly lower in the anesthesia control group than in control group. However, the liver vitamin E content was significantly (p < 0.05 andp < 0.01) increased in vitamin E, Se, and combination groups, whereas the vitamin A level in liver was not statistically different. In general, plasma levels of alanine aminotransferase, creatin kinase, total bilirubin, urea, red blood cell counts, packet cell volume, and hemoglobulin values were significantly (p < 0.05 andp < 0.001) increased during the anesthesia and returned to near control values after the vitamin E plus selenium injection. However, administration of vitamin E had less effect on the hematological and biochemical parameters compared to that of selenium and their combination with vitamin E. However, the white blood cell count and levels of alkaline phosphatase, aspartate aminotransferase, total cholesterol, triglycerides, total protein, and creatinine were not statistically influenced by the anesthesia. In conclusion, we observed that plasma levels of some enzymes and metabolites were significantly increased in the enflurane anesthesia of rats, whereas the liver vitamin E levels were slightly decreased. Therefore, we observed that vitamin E and selenium have a protective effect against anesthesia complication, but the effect of selenium appears to be much greater than the vitamin E.     

Inhibition of cytokinesis by asbestos and synthetic fibres

Using high-resolution timelapse microscopy, we have followed individual phagocytized fibres through the later stages of division in MeT-5A human mesothelial cells and LLC-MK(2)monkey epithelial cells. The fibres used were crocidolite and chrysotile asbestos, fibrous glass (MMVF), and refractory ceramic fibres (RCF). Long fibres (15-80 microm) trapped within the cleavage furrow can partially or completely block cytokinesis. Cells proceed in one of three ways: (1) eventual completion of cytokinesis; (2) incomplete cytokinesis, resulting in two cells joined by a fibre-containing intercellular channel; or (3) failure of cytokinesis, resulting in a binucleate or trinucleate cell. Two factors associated with fibre-induced bi/trinucleation are: (1) an initial association between the fibre and the forming daughter nuclei, which is sometimes lost over time, and (2) disintegration of the midbody. The studies suggest that delay of cytokinesis by interzonal fibres can result in bi/trinucleation through the loss of midbody/intercellular bridge proteins that are required for completion of cytokinesis.     

Genetic effects of crocidolite asbestos in Chinese hamster lung cells

Chinese hamster lung cells cultured in the presence of crocidolite asbestos displayed inhibition of cell growth. Cell death was directly associated with the phagocytosis of larger fibres as observed with the aid of trypan blue. Water soluble components of crocidolite did not appear to inhibit cell growth. Chromosomal aberrations were induced by the asbestos. The aberrations were confined mainly to structural aberrations--breaks and fragments. Electron-microscopic preparation indicated that asbestos was readily contained in phagosomes. Phagocytosed asbestos appeared to be a weak mutagen in its ability to induce gene mutation at the hypoxanthine-guanine phosphoribosyl transferase locus.     

Protective role of intraperitoneally administered vitamins C and E and selenium on the levels of lipid peroxidation in the lens of rats made diabetic with streptozotocin

The aim of this work was to determine the protective effects of intraperitoneally administered vitamins C and E and selenium on the lipid peroxidation (MDA), glutathione peroxidase (GSH-Px), reduced glutathione (rGSH) activities in the lens of rats induced diabetic with streptozotocin (STZ). Lenses in the diabetic control group had a slightly higher mean level of MDA compared with lenses of the vitamin E and selenium groups, although the mean levels of MDA were significantly lower in control, combination, and vitamin C groups than in the diabetic control group (p < 0.05 andp < 0.01). However, MDA levels were significantly lower in vitamin C, vitamin E, and combination groups than in controls (p < 0.01). The GSH-Px activities of lenses were significantly higher in vitamin C-, vitamin E- and selenium-injected groups than that in the diabetic control group (p < 0.01), whereas, the activity of GSH-Px was significantly lower in the diabetic control group than in the control group. In addition, the rGSH content was seen to decrease only in the vitamin C group compared to both control and diabetic control groups (p < 0.05). In conclusion, the results from these experiments indicate that vitamins C and E and selenium can protect the lens against oxidative damage, but the effect of vitamin C appears to be much greater than that of vitamin E and selenium.     

Micronucleus induction in mouse bone marrow by phenacetin administered intraperitoneally or orally

The extent and time course of induction of micronucleated polychromatic erythrocytes (MNPCEs) in mouse bone marrow were examined after administration of phenacetin as an insoluble suspension in olive oil by intraperitoneal injection (i.p.) or gastric intubation (p.o.) to 2 strains of mice, MS/Ae and CD-1, at doses up to 1200 mg/kg. The toxicity of phenacetin and the sensitivity of micronucleus induction differed in the 2 strains, but there was little difference in the extent of MNPCEs induced by the 2 administration routes.     

Apoptosis induced by crocidolite asbestos in human lung epithelial cells involves inactivation of Akt and MAPK pathways

Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways. A. Baldys, P. Pande contributed equally to this publication.     

Protective role of intraperitoneally administered vitamin E and selenium on the antioxidative defense mechanisms in rats with diabetes induced by streptozotocin

The aim of this work was to determine the protective effects of intraperitoneally administered vitamin E and selenium (as Na₂SeO₃, Se) on the lipid peroxidation as thiobarbituric acid reactive substances (TBARS) and vitamin E levels, glutathione peroxidase (GSH-Px), reduced glutathione (GSH) activities in the plasma, red blood cell (RBC), liver, and muscle of rats with streptozotocin-induced diabetes. Fifty adult male Wistar rats were used and all rats were randomly divided into five groups. The first group was used as a control and the second group as a diabetic control. A placebo was given to first and second groups by injection. The third group was intraperitoneally administered with vitamin E (20 mg over 24 h), the fourth group with Se (0.3 mg over 24 h), and the fifth group with vitamin E and Se combination (COM) (20 mg vitamin E + 0.3 mg Se over 24 h). This administration was done for 25 days and the TBARS, vitamin E, GSH-Px, GSH levels in the plasma, RBC, liver, and muscle samples were determined. The vitamin E level in the plasma and liver was significantly (p < 0.05) higher in the control than in the diabetic control group. Also, the TBARS levels in the RBC, liver, and muscle were significantly (p < 0.05) lower in the control than in the diabetic control group. However, GSH-Px and GSH activities in RBC, liver, and muscle were not statistically different between the control and the diabetic control groups. The vitamin E levels in plasma and liver (p < 0.01 and p < 0.001) and GSH-Px activities (p < 0.01, p < 0.001) in RBC were significantly higher in vitamin E, Se, and COM groups than in both control and diabetic control groups. However, the TBARS levels of RBC, muscle, and liver in vitamin E and Se administered groups were significantly (p < 0.05-p < 0.001, respectively) decreased. These results indicate that intraperitoneally administered vitamin E and Se have significant protective effects on the blood, liver, and muscle against oxidative damage of diabetes. The abstract of this study was presented in Physiological Research 48(Suppl. 1), S99 (1999).     

Transdiaphragmatic lymphatic transport of intraperitoneally administered marker in hamsters.

OBJECTIVE: To determine whether transdiaphragmatic transport in hamsters is similar to that described in other animals by examining transport of an intraperitoneally administered marker. METHODS: Monastral blue B suspension was administered intraperitoneally to 28 male Syrian hamsters (Mesocricetus auratus). Four hamsters each were euthanized 7, 15, and 30 min, and 1, 2, 3, and 24 h later. Specimens were examined microscopically for presence of marker. RESULTS: Marker was present in intrathoracic lymphatic vessels and cranial and caudal mediastinal lymph nodes by 7 min after its administration. The amount of marker in lymph nodes increased with time. The subcapsular distribution of marker was consistent with lymphatic transport. By 1 h after its administration, marker was present in the liver, spleen, bone marrow, and mesenteric and mandibular lymph nodes. Patterns of marker distribution in these tissues were consistent with hematogenous transport, but the amount of marker was considerably less than that in the intrathoracic lymph nodes at corresponding times. CONCLUSIONS: Particulates were most likely translocated from the hamster peritoneal cavity to intrathoracic lymph nodes via transdiaphragmatic lymphatic vessels. A portion of the translocated particulates entered the blood, where they were distributed to a variety of tissues within a short time.     

Mobilization of iron from crocidolite asbestos by certain chelators results in enhanced crocidolite-dependent oxygen consumption.

The reactivity of iron on crocidolite asbestos with dioxygen was determined and compared with iron mobilized from crocidolite. Ferrozine, a strong Fe(II) chelator, was used to demonstrate that iron on crocidolite was redox active. More Fe(II) was mobilized from crocidolite (1 mg/ml) by ferrozine anaerobically (11.2 nmol/mg crocidolite/h) than aerobically (6.6 nmol/mg/h) in 50 mM NaCl, pH 7.5, suggesting that Fe(II) on crocidolite reacts with O2 upon aqueous suspension. However, suspension of crocidolite in 50 mM NaCl, pH 7.5, did not result in a measurable amount of O2 consumption. The addition of reducing agents (1 mM) increased the amount of Fe(II) on crocidolite, and addition of ascorbate resulted in 0.4 nmol O2 consumed/mg crocidolite/min. Therefore, iron on crocidolite had limited redox activity in the presence of ascorbate. However, mobilization of iron from crocidolite increased its redox activity. Citrate, nitrilotriacetate (NTA), or EDTA (1 mM) mobilized 79, 32, or 58 microM iron, respectively, in preincubations up to 76 h, and increased O2 consumption upon addition of ascorbate to 2.8, 7.6, or 22.0 nmol O2 consumed/mg/min, respectively. This activity depended only upon the presence of a component(s) mobilized from crocidolite by the chelators. Pretreatment of crocidolite with the iron chelator desferrioxamine B (10 mM) inhibited O2 consumption. The results of the present study suggest that iron on or in crocidolite is responsible for the redox activity of crocidolite, but that mobilization of iron by chelators such as citrate, NTA, or EDTA greatly enhances its redox activity. Thus, iron mobilization from crocidolite in vivo by low-molecular-weight chelators may lead to the increased production of reactive oxygen species which may damage biomolecules, such as DNA.     

Generation of hydroxyl radical by crocidolite asbestos is proportional to surface [Fe3+].

Differences among fibrous silicates to effect injury in biological systems have been postulated to reflect oxidant generation by structural iron within the crystal lattice of amphiboles. Iron is also coordinated to the surface of all silicates in concentrations which depend on the density of acidic functional groups. We tested the hypothesis that oxidant generation by crocidolite is proportional to surface-complexed iron rather than variance in the lattice concentrations of this transition metal. Surface iron was quantified after its reduction to Fe2+ and chelation by citrate. Thiobarbituric acid (TBA) reactive products and dihydroxybenzoic acid products of salicylate were employed as indices of nonspecific oxidant and hydroxyl radical generation, respectively. Surface iron, TBA reactive products, and dihydroxybenzoic acid products all diminished after pretreatment of crocidolite with the metal chelator deferoxamine in concentrations varying from 0 to 250 mM. Inclusion of deferoxamine in the reaction mixture provided similar results of diminishing both TBA reactive products and dihydroxybenzoic acid generation. We conclude that oxidant generation by crocidolite is proportional to surface concentrations of iron which can be chelated using deferoxamine. The design of synthetic fibers without health effects after exposure will likely necessitate decreasing the number of surface acidic functional groups to diminish the capacity to complex iron (i.e., minimize the percentage SiO2).     

Determination of 8-hydroxydeoxyguanosine by an immunoaffinity chromatography-monoclonal antibody-based ELISA

The postulated importance of oxidative damage to DNA in aging and age-related degenerative pathologies such as cancer has prompted efforts to develop sensitive quantitation methods. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely used marker for oxidative damage to DNA. To develop an immunoassay for quantitation of 8-OHdG, two monoclonal antibodies have been developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 1F7 has 50% inhibition at 5 pmol 8-OHdG and 1 × 105 pmol dG, while antibody IF11 has 50% inhibition at 2.5 pmol 8-OHdG and 2000 pmol dG. Both antisera crossreact with guanosine and several structurally related derivatives, including 6-and 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanine, and 7-methylguanosine. Immunoaffinity columns were prepared with antibody 1F7, which exhibits higher selectivity than 1F11, to isolate 8-OHdG from DNA hydrolyzates followed by ELISA quantitation with antibody 1F11. This method allows the analysis of approximately one 8-OHdG/105 dG using 100μg DNA. To validate the assay, DNA extracted from human placental tissues were assayed by both ELISA and HPLC with electrochemical detection. Values by both methods correlated well (r = 0.87, p < 0.001), but the levels determined by ELISA were approximately sixfold higher than those determined by HPLC. This may be due to oligonucleotides detected by the ELISA but not the HPLC method or crossreactivity with other damaged bases present in the immunoaffinity purified material. Placental samples from current smokers had significantly higher 8-OHdG by ELISA than those from nonsmokers (p < 0.05). The method of immunoaffinity purification combined with ELISA quantitation has sufficient sensitivity for detecting 8-OHdG in human DNA samples. Although absolute values are higher than those determined by HPLC, the method provides a good alternative to the HPLC-EC method for monitoring relative oxidative damage in molecular epidemiological studies.     

Photosensitized formation of 8-hydroxydeoxyguanosine in cellular DNA by riboflavin.

Photosensitized formation of 8-hydroxydeoxyguanosine (oh8dG) in DNA by riboflavin has recently been shown in vitro. The present study describes the formation of oh8dG in cellular DNA by photo-irradiation of cultured mammalian cells in the presence of riboflavin. Formation of oh8dG was dependent on the concentration of riboflavin as well as the duration of photoirradiation. These results suggest that photosensitized formation of oh8dG in DNA by riboflavin may be involved in photocarcinogenesis.     

Induction of early alveolar injury by inhaled asbestos and silica

Inhaled asbestos fibers and silica crystals are known to cause interstitial fibrotic lung disease in animals and humans. The initial cellular events and biochemical mechanisms that lead to development of disease are poorly understood. In ongoing studies reviewed here it has been shown that inhaled particulates small enough to pass through the conducting airways are deposited initially at the bifurcations of alveolar ducts. Within hours after brief exposure, alveolar epithelial cells phagocytose inhaled particles that subsequently are translocated to interstitial matrix and fibroblasts. Within 48 h after exposure, inhaled asbestos on alveolar surfaces activates a complement-dependent chemotactic factor for macrophages that accumulate at duct bifurcations. Epithelial cells, macrophages, fibroblasts, and the interstitial matrix are significantly altered by brief (1- 5-h) exposure to chrysotile asbestos. The basic mechanisms that mediate these initial events remain to be defined.     

Induction of angiogenesis by intraperitoneal injection of asbestos fibers

Tumors and activated macrophages release angiogenic factors that stimulate migration and proliferation of capillaries. We studied the development of angiogenesis before the appearance of mesotheliomas in C57B1/6 mice. Weekly i.p. injections of crocidolite asbestos fibers produced mesotheliomas after 30-50 wk. The initial histologic response to asbestos fibers was a nodular lesion on the peritoneal lining composed of clusters of fibers, activated macrophages, and proliferating mesenchymal cells. The earliest visible evidence of angiogenesis was seen surrounding 7% of these lesions 14 days after a single injection of 200 micrograms of crocidolite asbestos fibers. After six weekly injections, 30% of the lesions containing asbestos fibers were surrounded by a capillary network radiating toward the center of the lesion. Other mineral fibers, including chrysotile asbestos and fiberglass, also induced angiogenesis after six weekly injections. In contrast, only 8% of the lesions containing short asbestos fibers (90.6% less than or equal to 2.0 microns) and 9% of the lesions containing silica particles showed evidence of angiogenesis. We conclude that tumorigenic mineral fibers induce angiogenesis in the peritoneal lining, whereas nontumorigenic mineral particles or short asbestos fibers are less effective. Ingrowth of new blood vessels around clusters of asbestos fibers may facilitate the later emergence of mesotheliomas at these sites.     

不同微生物大鼠腹腔注射模型尿液蛋白组学的比较

不同的微生物都可以引起腹腔感染,文中尝试利用尿液来区分不同的微生物感染.通过在大鼠腹腔内分别注射大肠杆菌、金黄色葡萄球菌和白色念球菌建立3种模型,收集感染后0、12、36、72h的尿液,并使用液相色谱串联质谱技术(LC-MS/MS)对尿蛋白进行分析.与感染前相比,在大肠杆菌腹腔注射模型中共鉴定到69个差异蛋白,在金黄色...