Iron overload induces apoptosis of murine preosteoblast cells via ROS and inhibition of AKT pathway.

OBJECTIVE: This study investigates the inhibitory effect of iron overload on MC3T3-E1 cells and its molecular mechanism. METHODS: MC3T3-E1 cells were grown under different concentrations of FAC (ferric ammonium citrate), and the WST-8 assay was used to investigate the proliferation of cells following FAC with or without deferasirox. DCFH-DA fluorescence probe was applied to detect the cellular reactive oxygen species (ROS) level. The apoptotic cells were analyzed with Annexin V-FITC/PI and the Hoechst 33258 nuclear staining assay. The JC-1 staining assay was applied to observe the change in the mitochondrial transmembrane potential. The expression levels of caspase-3, PARP, Bcl-2 family proteins, and AKT kinase were detected with the Western blot assay. RESULTS: Iron overload had a cytotoxic effect on MC3T3-E1 cells in a dosage-dependent way and resulted in increasing level of intracellular ROS. Iron overload induced apoptosis in MC3T3-E1 cells via a caspase-dependent mechanism that is accompanied by mitochondria dysfunction and decreased expression of anti-apoptotic proteins. The expression levels of cleaved-caspase-3 and cleaved-PARP were upregulated, while the expression levels of caspase-9, caspase-7, caspase-3, and PARP were downregulated. Phosphorylation of AKT kinase decreased. CONCLUSION: Iron overload can generate ROS in cells, inhibit AKT kinase and its downstream proteins activity, and subsequently initiate apoptotic events.

Uptake of oxytetracycline and its phytotoxicity to alfalfa (Medicago sativa L.).

A series of experiments were conducted in a hydroponic system to investigate the uptake of oxytetracycline (OTC) and its toxicity to alfalfa (Medicago sativa L.). OTC inhibited alfalfa shoot and root growth by up to 61% and 85%, respectively. The kinetics of OTC uptake could be well described by Michaelis-Menten equation with Vmax of 2.25 micromol g-1 fresh weight h-1, and Km of 0.036 mM. The uptake of OTC by alfalfa was strongly inhibited by the metabolic inhibitor, 2,4-DNP (2,4-dinitrophenol), at pH 3.5 and 6.0, but not by the aquaporin competitors, glycerol and Ag+. OTC uptake, however, was significantly inhibited by Hg2+, suggesting that the inhibition of influx was due to general cellular stress rather than the specific action of Hg2+ on aquaporins. Results from the present study suggested that OTC uptake into alfalfa is an energy-dependent process.

The veterinary antibiotic oxytetracycline and Cu influence functional diversity of the soil microbial community.

There are increasing concerns over the effects of veterinary antibiotics and heavy metals in agricultural soils. The widely used veterinary antibiotic oxytetracycline (OTC), Cu and their combination on soil microbial community function were assessed with the Biolog method. The microbial community was extracted from the soil and exposed to a 0.85% sodium chloride solution containing OTC (0, 1, 5, 11, 43, 109 and 217 microM), or Cu (0, 10, 20, 100 and 300 microM), or combination of the two pollutants (OTC 0, 5, 11 microM and Cu 0, 20 microM). Functional diversity, evenness, average well color development (AWCD) and substrate utilization decreased significantly with increasing concentrations of OTC or Cu (p < 0.005). The critical concentrations were 11 microM for OTC and 20 microM for Cu. The combination of OTC and Cu significantly decreased Shannon's diversity, evenness and utilization of carbohydrates and carboxylic acids compared to individual one of the contaminants. The antibiotic OTC and Cu had significant negative effects on soil microbial community function, particularly when both pollutants were present.