Caco-2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cells are normally cultured in the presence of 15-20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco-2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush-border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, gamma-glutamyltransferase, aminopeptidase N, and dipeptidyl-dipeptidase IV), although the levels of sucrase activity were lower in ITS-supplemented medium. Coating petridishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush-border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 x 10(-8) M) was added to the ITS-supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while gamma-glutamyltransferase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 x 10(-6) M). On the other hand, hydrocortisone (HC, 10(-6) M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco-2 cells can be maintained in serum-free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.
The decline in the population of pollinators is a worrying phenomenon worldwide. In North America, the extensive use of herbicides in maize and soya crops may affect the health of nontarget organisms like the honey bee. In this study, caged honey bees were exposed to realistic doses of atrazine, metolachlor, and glyphosate for 10 days via contaminated syrup. Peroxidation of lipids was evaluated using the thiobarbituric acid reactive substance (TBARS) test, and diet-derived antioxidants-carotenoids, all-trans-retinol (at-ROH) and α-tocopherol-were detected and quantified using reversed-phase HPLC techniques. Significant increases in syrup consumption were observed in honey bees exposed to metolachlor, and a lower TBARS value was recorded for the highest dose. No relationship was observed between the peroxidation of lipids and the levels of antioxidants. However, β-carotene, which was found to be the most abundant carotenoid, and at-ROH (derived from β-carotene) both decreased with increasing doses of atrazine and glyphosate. In contrast, metolachlor increased levels of at-ROH without any effects on β-carotene. These results show that the honey bee carotenoid-retinoid system may be altered by sublethal field-realistic doses of herbicides.
Chemical Speciation and Bioavailability (2001), 13(1) ABSTRACTThe bioavailability and toxicity of a dissolved metal are closely linked to the metal's chemical speciation in solution. A variety of inorganic and organic ligands are often used in laboratory toxicity tests to control the concentration of labile trace metal in solution. Computerised chemical speciation models based on thermodynamic principles can be used to estimate metal speciation under such experimental conditions. However, these models are sensitive to the quality of their thermodynamic databases. Detailed protocols for the incorporation of reliable equilibrium formation constants into widely available computer chemical speciation programs (e.g., MINEQL+ and MINTEQ) are provided. The examples demonstrate both the benefits and the potential pitfalls involved in the use of chemical speciation models. The application of chemical speciation modelling to metal toxicity studies is discussed and guidelines are proposed for its proper use. Both defined media and chemical speciation programs have co-existed for two decades but the combined use of these techniques has been reserved for those possessing in-depth knowledge of both chemistry and biology. The techniques presented should enable an investigator with basic biological, chemical and computing skills to design an aqueous medium and incorporate correct thermodynamic constants into a computer chemical speciation program, starting from a standardised database, thereby providing a sound framework for critically assessing the biological response of a particular test organism to a given metal.
109Cd uptake was studied using the highly differentiated TC7 clone of Caco-2 cells as a model of human enterocyte function. Intracellular accumulation of 0.3 microM 109Cd involved a rapid and a slow uptake phase, which resulted in complete equilibration (t(1/2) = 17.3 +/- 1.3 min) with an apparent in-to-out distribution ratio (alphae) of 11.6 +/- 0.8. The amplitude of the rapid phase (U0) and the rate of the slow phase (V) were similarly reduced in the less differentiated PF11 clone, but comparable alphae values were observed at equilibrium. In both clones, the t(1/2) and alphae values increased and decreased, respectively, upon addition of unlabeled Cd to the uptake media. In TC7 cells, 109Cd uptake at 1 min (U1) was unaffected by Ca concentrations four order of magnitude in excess, but both U0 and V demonstrated similar sensitivities to unlabeled Cd, Zn and sulfhydryl-reactive agents. Only U0 disappeared when EDTA was present in the wash solutions. U1 showed saturation kinetics and the data were found compatible with a model assuming rapid initial Cd binding and transport through a unique transport protein (Km = 3.8 +/- 0.7 microM). Cd efflux kinetics demonstrated partial reversibility in EDTA-containing solutions, suggesting that the taken up Cd might be both tightly and loosely bound to intracellular binding sites. However, the displacement of 109Cd measured at 65 min failed to reveal this heterogeneity: the data were found compatible with a model equation assuming the presence of one class of high-capacity high-affinity binding sites. We conclude that a slow-transport fast-intracellular binding mechanism of Cd uptake best accounts for these results and that Cd transport most likely involves a carrier-type of protein unrelated to Ca absorption.
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