Toxicological Biomarkers Of Organoarsenical Feed Additives In Environment Based On Tetrahymena Thermophila Model

Organoarsenicals including roxarsone (ROX) or arsanilic acid (PASA)are commonly used as feed additives in poultry operation. However90%ofthese organoarsenical feed additives could be released into natural ecologicalsystems of soil and water through animal feces and urine without chemicalchange. Subsequently the organoarsenicals may be degraded into high acutetoxic inorganic arsenic by means of the poultry litter land application process,under a series of complicated environmental conditions such as sunlight,microbological decomposition et al. It poses as a latent hazard to humanhealth due to arsenic accumulation through the ecological food cycle.It is well known that Tetrahymena thermophila play a key role withinthe aquatic microbial loop as an unicellular protozoa and is sensitive to toxicmaterials in aquatic environment. Additionally Tetrahymena thermophila hasits inherent characteristics as below: it can be multiplied rapidly; it can beeasily cultured in lab of low cost. Therefore Tetrahymena thermophila hasbeen deemed as an ideal organism model in field of environmental toxicological research.Here the environmental toxicological biomarkers of organoarsenicalfeed additives (ROX or PASA) and their degradation mixtures were studiedbased on Tetrahymena thermophila model. In our works, hydridegeneration-atomic fluorescent spectrometry (HG-AFS), protein gelelectrophoresis and biological mass spectrometry were utilized as keyanalytical techniques.Firstly, it showed that total arsenic uptake on cell membrane and in theintra-cellular of tetrahymena can be environmental biomarkers oforganoarsenicals feed additives and their degradations mixtures. The resultsindicated that: total arsenic uptake of Tetrahymena thermophila exposed toorganoarsenicals in original chemical forms mostly took place on cellmembrane. However total arsenic uptake of cells exposed to simulateddegradations of organoarsenicals (ROX or PASA、As(Ⅲ) and As(Ⅴ) mixedwith different ratios) increased obviously and most accumulated in theintra-cellular, which was similar to that of Tetrahymena thermophila exposedto inorganic arsenic. Furtherly silica gel carrier was coated with lipidsome tosimulate cell membrane of Tetrahymena thermophila. It was used to clarifyfor the mechanism on different uptake of arsenic in Tetrahymena thermophilaafter exposed to organoarsenicals (ROX or PASA) and their degradation mixtures respectively.Secondly specific protein distribution of Tetrahymena thermophilaexposed to organoarsenicals (ROX or PASA) and their degradation mixturesas environmental biomarkers were studied in our work. It indicated that: therewere not definite differences on total protein distribution for whole cell ofTetrahymena thermophila exposed to organoarsenicals in original chemicalforms (ROX or PASA), in comparion to the cultured control cell samples.However the proteins band with molecular masses around19kDa～25kDawas observed after the Tetrahymena cell exposed to degradation mixtures oforganoarsenicals, which was similar to the results of Tetrahymena cellexposed to inorganic arsenic. Nano materials were used to preferentiallyenrich and separate protein extraction from intra Tetrahymena thermophila.Furtherly, a protein band with molecular masses around25kDa～35kDa wasobserved in the protein gel separation results, after the extraction ofintra-cellular Tetrahymena was selectively enriched by functional nano-sizedmaterials (Here Tetrahymena cells were exposed to both organoarsenicals inoriginal chemical forms and the control cell samples). But the protein bandaround25kDa～35kDa disappeared for Tetrahymena cells exposed toorganoarsenicals degradation mixtures, which was similar to the results afterTetrahymena cell exposed to inorganic arsenic. The biological mass spectrometry results showed that: the specific protein band with molecularmass around25kDa～35kDa were14-3-3, and the western blotting resultsconfirmed that this protein band was positive to phosphorated proteinantibodies. It is known protein14-3-3plays an important role during cellsignal transduction process by interaction with targeted proteins.