In vitro effect of calcium dobesilate on oxidative/inflammatory stress in human varicose veins.

AIM: To determine whether calcium dobesilate can act in chronic venous insufficiency by similar antioxidant, anti-inflammatory mechanisms as in diabetic retinopathy. METHODS: Calcium dobesilate was tested in vitro for its protective action against oxidative/inflammatory stress in human varicose veins. Varicose greater saphenous veins were obtained from 14 patients (11 men, 3 women) aged 53-65 years. Oxidative stress was induced exogenously in the vein segments, with the phenazine methosulphate (PMS)/NADH couple. Total antioxidant status (TAS) and malondialdehyde (MDA) contents were used as markers of oxidative stress. RESULTS: Calcium dobesilate significantly prevented oxidative disturbances in the micromolar range. PMS/NADH-dependent TAS decrease was fully prevented with IC(50) = 11.4 ± 2.3 µmol/L (n = 6 veins), whereas MDA increase was fully prevented with IC(50) = (102 ± -3) µmol/L (n = 6 veins). Calcium dobesilate acted quali- and quantitatively like rutin, the reference compound. Comparison with pharmacokinetic data suggests that calcium dobesilate can act at therapeutic concentrations. CONCLUSION: Calcium dobesilate protected human varicose veins against oxidative stress in vitro at levels that correspond to therapeutic concentrations. Further studies are required to investigate whether a similar action is found in varicose veins from patients orally treated with calcium dobesilate.

Effect of bile type on the bioaccessibility of soil contaminants in an in vitro digestion model.

Soil ingestion is an important pathway of exposure for many nonvolatile contaminants for man and in particular for children. A fraction of the ingested contaminant may not dissociate from the soil particles during digestion in the gastrointestinal tract, and is thus not available for transport across the intestinal epithelium. In order to estimate the contaminant fraction that is mobilized from soil, i.e., the bioaccessible fraction, several in vitro digestion models have been developed. The currently existing digestion models display many differences. One aspect that may affect bioaccessibility and may induce differences between digestion models is the bile that is used. Often freeze-dried bile of animal origin is preferred to purified bile salts. However, also the animal origin of bile may give rise to differences in bioaccessibility because bile composition appears to be species dependent. In the present study, we compared the bioaccessibility of benzo[a]pyrene, arsenic, cadmium, and lead of four different soils after digestion with ox bile from two different suppliers, pig bile, and chicken bile. Bioaccessibility appeared to vary amongst the different soils and contaminants. Only chicken bile increased the bioaccessibility of lead and cadmium significantly and relevantly for one of four soils. For chicken bile, the bioaccessibility of lead was 3-5.5 times greater than for the other bile types and the bioaccessibility of cadmium was 1.5 times greater. In all other cases, the bioaccessibility differences were less than 10%, which is considered irrelevant for risk assessment purposes.

Development of an in vitro digestion model for estimating the bioaccessibility of soil contaminants.

Soil ingestion can be a major route of human exposure to many immobile soil contaminants. The present risk assessment is based on toxicity studies in which contaminants are typically ingested in liquid or food matrices. The difference in bioavailability of contaminants ingested in a soil matrix is not taken into account. To become bioavailable, contaminants first need to become bioaccessible, i.e., they must be mobilized from the soil during digestion. Soil contaminants may be less bioaccessible than contaminants from liquid or food, so that the risks can be overestimated. This article describes the development of an in vitro human digestion model that is physiologically based. It can be used as a tool to assess bioaccessibility. We explain the rationale behind the experimental design of the model. We address the aspects of the simulated compartments of the gastrointestinal tract, temperature, soil-to-fluid ratio, ratio of digestive juices, transit times, centrifugation, pH values, mixing, constituents and their concentrations, and bile. The optimized in vitro digestion model was applied in a case study. The bioaccessibility of lead in pottery flakes with glazing was determined and compared to the bioaccessibility of lead in the soil from which the pottery flakes were removed. The data indicate that pottery flake lead is considerably less bioaccessible (0.3 +/- 0.2%) than lead in soil without pottery flakes (42-66% at the same site, and 28-73% at other sites in the same town). Furthermore, bioaccessibility values of lead in soil appear to be less than calculated bioaccessibility values for dietary lead (which are based on the criterion used by the Dutch risk assessment and on literature absorption data). This indicates that accounting for the matrix of ingestion can affect the exposure assessment for lead. The in vitro digestion model is a promising tool for studying the effect of the ingestion matrix on bioaccessibility.

Eimeria tenella: hsp70 expression during sporogony.

There is an increasing interest in identifying the parasite components involved in the maturation, development, and infectivity of intracellular protozoan parasites. In the present study, a heat shock protein (hsp) of the family of 70 kDa hsp (hsp70), which play important roles in the stage conversion and virulence of these parasites, was examined. Whereas hsp70 expression has been examined in Eimeria tenella within host tissues, in the present study, oocysts of E. tenella were used to investigate the expression of hsp70 during sporulation without interference from the host; hsp70 expression during excystation was induced by incubating sporulated oocysts under various experimental conditions to produce the stimuli necessary for sporozoites to become active and to excyst in vitro. Hsp70 was detected by immunohistochemical techniques; quantitative flow cytometric analysis was also been carried out using specific monoclonal antibodies against hsp70. Hsp70 was expressed during sporulation but was not found in sporulated oocysts after the completion of sporulation. Oocysts re-expressed hsp70 when excystation was induced. The presence of hsp70 prior to infection may preadapt the parasite for additional stress in the host and may be involved in the formation of sporozoites.

Antioxidant intracellular activity of genistein and equol.

Antioxidant effects of isoflavonoids have recently been described. To learn whether the isoflavonoids genistein and equol have actions on the intracellular free radicals, human neutrophils and J774 monocyte-macrophage cell line were used to measure the intracellular production of O(2) (superoxide anion) and H(2)O(2) (hydrogen peroxide) by flow cytometry. The results shown significatives decrease in O(2) and H(2)O(2) production after 1 hour of incubation with equol and genistein. The phagcytic oxidant production decreased owing to the effects of both isoflavonoids in a concentration-dependent manner.

Flow cytometry study of the role of superoxide anion and hydrogen peroxide in cellular photodestruction with 5-aminolevulinic acid-induced protoporphyrin IX.

Flow cytometry was used to investigate the participation of reactive oxygen species, other than singlet oxygen, in the cytotoxic effect of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in vitro in A-431 squamous cell carcinoma (SCC) cells and human skin fibroblasts (HSF). We used propidium iodide to determine cellular cytotoxicity, hydroethidine to measure intracellular superoxide anion (O2-) and dihydrorhodamine 123 to assess intracellular hydrogen peroxide (H2O2) content. Our data support the importance of the incubation time with ALA in the selectivity of PDT with ALA against SCC cells, inducing minimum damage on normal HSF. Photoradiation mortality curves of the response of these cell lines to ALA-induced PpIX photosensitization correlated with the extent of photosensitizer accumulation. Intracellular O2- production correlated with cell death, increasing both in a light dose-dependent fashion in ALA treated cells. This correlation was not observed with H2O2-intracellular production. These results suggest the effectiveness of PDT with ALA in vitro in SCC, the significant participation of O2- in its phototoxic mechanism, and the usefulness of flow cytometry in the study of the cytotoxic effect of ALA-induced PpIX PDT.

Superoxide anions produced by inflammatory cells play an important part in the pathogenesis of acid and pepsin induced oesophagitis in rabbits.

BACKGROUND: Reactive oxygen metabolites have been associated with gastrointestinal injury. OBJECTIVE: To investigate whether mucosal reactive oxygen metabolites are involved in acid and pepsin induced oesophagitis, and if so, which specific metabolites. METHODS: The effects of free radical scavengers and the anti-inflammatory drug ketotifen on rabbit oesophagitis induced by acidified pepsin were studied. Isolated oesophageal cells were obtained before and after oesophageal injury and the generation of superoxide anion and hydrogen peroxide was analysed by flow cytometry. The presence of inflammatory cells was determined by indirect immunofluorescence with a mouse antirabbit CD11b antibody. RESULTS: Of the free radical scavengers tested, superoxide dismutase, which reacts with the superoxide anion, significantly reduced oesophagitis, whereas catalase, which reacts with hydrogen peroxide, had only a mild effect and dimethylsulphoxide had no effect. Ketotifen significantly reduced the inflammation and also prevented the induction of oesophagitis. Isolated cells obtained from the oesophageal mucosa after acidified pepsin exposure generated increased amounts of superoxide anions, which were mainly produced by CD11b positive cells. CONCLUSIONS: Reactive oxygen metabolites, especially superoxide anion, produced by inflammatory cells play a significant part in the genesis of oesophagitis induced by acid and pepsin in rabbits and might be a target for future medical therapy.