Study On Toxic Effects Of Organic Phase On Taxus Cuspidata In Two- Phase Cultures

The molecular toxicity of organic solvents (oleic acid, dibutyl phthalate) on Taxuscuspidata cells was investigated in two-liquid phase suspension cultures from thepoint of cell metabolism, cell-membrane permeability, genetic stability, redox statepotentiality, proteome and it can help us understand the mechanism of two-liquidphase suspension cultures, suitable organic solvents selection, which is the focus ofthis work. The main results as follows: The effects of organic solvents (oleic acid, dibutyl phthalate) on cell membrane of T.cuspidata, including cell-membrane injury and surface topography of the protoplastmembrane were investigated in two-liquid phase cultures. The results showed thatelectrical conductivity and concentration of malonyl dialdehyde varied significantlywhen the amounts of oleic acid & dibutyl phthalate were 6% (v/v) or more, but theydid not change obviously when the amounts of oleic acid & dibutyl phthalate were 2%(v/v). Analysis of the surface topography protoplast membrane with EnvironmentalScanning Electron Microscope showed that organic solvents partly damaged theplasmodesmata and formed tinny holes, and so possibly changed the membranepermeability of T. cuspidata. Results of the detecting of Inductively Coupled PlasmaOptical Emission Spectroscopy (ICP-AES) showed that the addition of organicsolvents activated the influx of Ca2+ influx in a short time, K+ efflux coupled with H+influx. This work showed that the organic solvents severely affected the cellmembrane integrity, leading to an increase in membrane permeability for thesecondary metabolites in two-liquid-phase cultures of T. cuspidata. The addition of organic solvents decreased cell mitosis and interfered with thebalance between apoptotic and mitotic index. The occurrence of some abnormal cellsfurther confirmed that organic solvents induced an abnormal mitosis of T. cuspidatacells. And there was an inverse correlation between mitotic index and apoptotic index. Generation of active oxidative species (AOS) induced by organic solvents intwo-liquid phase suspension cultures of T. cuspidata was investigated. It was foundthat T. cuspidata cells respond to oleic acid with oxidative bursts. Their triphasiccharacteristics were almost similar in both intracellular H2O2 production andextra-cellular production. Additionally, inhibition studies with diphenyleneiodonium suggested that the key enzyme responsible for oxidative bursts in two-liquidphase suspension cultures is primarily NADPH oxidase. Investigation of the IIrelationship between active oxidative species and defense responses induced byorganic solvents indicated that 4% (v/v) organic solvents increased the levels ofantioxidant enzymes of SOD, POD and CAT and antioxidant capacity of AsA andGSH, and the cells presented a positive endogenous protection effect. However, whenthe content of organic solvents reached a critical value (6%, v/v) no further increase inantioxidant enzymes and antioxidant capacity were observed. This indicates that thedefense responses can make a role in a certain range. Beyond the certain range, theoverall AOS scavenging machinery was not induced.