Analysis Of Nanomaterials Toxicity Based On A Stress-responsive Whole-cell Bacterial Biosensor

With the unique properties, nano-materials nowadays have been applicated in a wide range of fields. Meanwhile, their potential negative impacts on environment and human health have also attracted great concern among the public. However, compared to the rapid development of the synthesis technology and broad application, the risk estimation of using nanomatterials to human health and the environment has not yet been fully established. Moreover, due to the extensive use of nano-materials, they could easily interact with pollutants in the environment—especially persistent organic pollutants (POPs), due to their recalcitrance and widely existence in the environment. Toxicity of POPs in the environment or biological systems is widely investigated, while the researches about the synergistic toxicity between nano-materials and POPs have never been reported yet. Therefore, we investigated the toxic effects and the primary toxic mechanisms of a typic nanomaterial, copper nanoparticles (CuNPs), and the synergistic toxicity between CuNPs and Pentachlorophenol (PCP) based on a stress-responsive whole-cell bacterial biosensor, which is composed with a panel of recombinant bacteria, and is able to respond to several classes of threats (stresses) with signal production. The main points of this thesis are summarized as follows:1. The toxicological effects and the source of toxicity of CuNPs are investigated based on a stress-responsive whole-cell bacterial biosensor (a panel of recombinant bioluminescent bacterial which specifically respond to oxidative stress, protein damage, membrane damage and DNA damage). According to the responses of the biosensing strains, it is found that CuNPs induce not only oxidative stress in E. coli, but also protein damage, DNA damage, and cell membrane damage, and ultimately cause cell growth inhibition. Through enzyme detoxification analysis, the toxicological effects of CuNPs are traced to H2O2generation from CuNPs. The soluble copper of CuNPs in the cell culture was detected by AAS, and the production of Cu(Ⅰ) from CuNPs was further checked by2,9-dimethyl-1,10-phenanthroline (neocuproine, Nc). With the help of cuprous chelator (THPTA), we found that the oxidation of the released Cu (Ⅰ) has close relation to H2O2production. In addition, TEM study showed that CuNPs can be adsorbed and incepted fast by the cells. Comparatively, copper microparticles are relatively stable in the system and practically non-toxic, which indicates the importance of toxic estimation of materials at nanoscale. In addition, Cu (Ⅱ) ion can induce protein damage, membrane damage, and slight DNA damage at only relatively high concentration. The current study not only reveals that the biological toxicity of the copper particles is particle size dependent, but also the valence state of metal ion analysis in the study of toxicity of metal nanomaterials is very necessary.the preliminary mechanism of toxicity of CuNPs, and suggests that the stress-responsive whole-cell bacterial biosensor can be used as a simple and promising tool for rapid screening in vitro toxicity of nanoparticles and studying the primary mechanism of the toxicity.2. The synergistic toxicity between CuNPs and PCP are investigated by the whole-cell biosensor strains. It is found that co-existent of PCP in the medium resulted in largely increased luminescent signal of the cells, which are sensitive to oxidative stress and DNA damage, to CuNPs, which indicates that co-existense of CuNPs and PCP resulted in synergistic oxidative stress and DNA damage to the bacteria. The enzyme detoxification analysis, as well as toxic analysis of the co-existent toxicity between PCP and Cu (I) or H2O2, suggested that the synergistic toxicity between CuNPs and PCP was related to Cu (I) and H2O2generated by CuNPs in the cell medium. LDH measurements show that both CuNPs and PCP can cause the cell membrane damage, and the co-existence of CuNPs and PCP aggravate the cell membrane damage. Therefore, the enhanced permeability of cell membrane caused by PCP makes CuNPs easier to enter the cell, resulting in the increase of cytotoxicity. In addition, the synergistic toxicity of CuNPs and PCP probably has no relationship with the PCP metabolites based on HPLC anlysis.