Establishment And Primary AppJication Of The Integrated Discrete Multiple Human Originated Organ Cells Co-culture System

With the release of the landmark report entitled―Toxicity Testing in the21st Century:A Vision and Strategy‖by the U.S. National Academy of Science (NAS), precipitated arevolutionary change in the way of toxicity testing would be conducted. Targeted testingand pathway testing based on human cells will replace the traditional toxicity testingmethod in animals, and will become a hot area of research and developing trends oftoxicity testing. U.S. Environmental Protection Agency (EPA) and National ToxicologyProgram (NTP), and other agencies have sponsored and conducted several researchprojects. The current studies are expanded from three angles: First, try to find the basisfor targeted testing via comparing the toxic sensitivity of different human cell lines;Second, toxicity pathway assays of different chemicals; Third, research methods fortoxicity pathway and data assay. The current project will mainly focus on targeted testingresearch.On the targeted testing, using cells from possible toxicity target-organs or comparingsensibility response to toxicants of multiple cells are the common approaches now. Thedeficiencies of these methods include: First, target-organs are unknown for sometoxicants; Second, target organs could not be detected rapidly and accurately as cells arecultured individually, not under the same condition, and could not imitate the in-vivoenvironment although several cells/organs are involved; Third, numerous toxicant couldnot be activated or detoxicated unless metabolized via liver or other organs. And theutility of S9fractions as metabolism system could not imitate the actual process in-vivo.In addition, the in-vitro individual cell culture is unable to exhibit the intercellularinteractions in the microenvironment in-vivo. In general, the current testing methodscould not meet the targeted testing. By this, our research approach is to establish a theintegrated discrete multiple organ co-culture model using human hepatic cell lines and celllines from other major target organs for toxicity.Compared with micro-scale materials, many unprecedented biological effects andcharacters are noted in nanometer materials due to their special physicochemical properties,which bring new challenges for toxicity safety evaluation. Fully discover their new ordifferent toxic effects from micro scale materials, and find their toxic characters are thepivotal issues for safety evaluation of nanometer materials. Investigating the toxicitytarget organs of nanometer materials and compared with micro scale materials may will provide some scientific basis for the safety evaluation of nanometer materials.The objectives of the current study are to establish a novel in vitro model of integrateddiscrete multiple organ co-culture using human originated cells from different cell lines; todetect the possibility whether this model can be used for toxicity target-organs screening ofexogenous toxicants; to evaluate the metabolic abilities of cell lines sourced from liver;and to find targeting effects of nanometer materials via using the established model.1. To establis h a novel in vitro model of integrated discrete multiple organco-culture of human originated cells from different cell linesSelected representative cell lines commonly used in toxicity target organ testing,including pulmonary (A549, MRC-5), Hepatic (HepG-2), neural system (SH-SY5Y),nephritic (HEK-293), and cardiac (CCC-HEH-2). Integrated of adjacent5wells bypunching holes in each well wall using commercial48-well plates, and these integratedwells are considered as a unit. Media in each well could be interconnected via the holesin the wall. Comparing growth of each cell when they were cultured separately orcultured in the plate as described above. Also, the growth of cells cultured in DMEM andin recommended media by ATCC.Result: All cells grew well in DMEM with exception of MRC-5. And pulmonary(A549), Hepatic (HepG-2), neural system (SH-SY5Y), nephritic (HEK-293), and cardiac(CCC-HEH-2) cultured in DMEM were selected to establish the integrated discretemultiple organ co-culture (IdMOC) model. The cell growth curves of all5cells of singletype of cell culture and co-culture were coincident. Indicated, the integrated discretemultiple organ co-culture model was established successfully.2. To investigate the possibility of IdMOC used for toxicity target-organscreeningTo detect the target-organ effects of the above5cell lines after exposed to knowntoxicants (carbon tetrachloride, sodium fluoride, hydroxydopamine, streptomycin sulfate,doxorubicin hydrochloride, paraquat, busulfan, and cadmium chloride) for24/72hoursusing MTT method. The toxicity effects to different cells are listed below (exposed for24hours): carbon tetrachloride: HepG-2> HEK-293> CCC-HEH-2> SH-SY5Y(based on IC50and IC10values);2) sodium fluoride: HEK-293> CCC-HEH-2>HepG-2> A549> SH-SY5Y (based on IC50and IC10values);3) hydroxydopamine:SH-SY5Y> CCC-HEH-2> HEK-293> A549> HepG-2(based on IC50values), SH-SY5Y> A549> HEK-293> CCC-HEH-2> HepG-2(based on IC10values);4)streptomycin sulfate: SH-SY5Y> HEK-293> CCC-HEH-2> HepG-2> A549(based onIC50values), HEK-293> SH-SY5Y> CCC-HEH-2> HepG-2> A549(based on IC10values);5) doxorubicin hydrochloride: HepG-2> HEK-293> SH-SY5Y> CCC-HEH-2(based on IC50values), CCC-HEH-2> HEK-293> HepG-2> SH-SY5Y> A549(based onIC10values);6) paraquat: HepG-2> HEK-293> SH-SY5Y> A549> CCC-HEH-2（based on IC50values），HepG-2> HEK-293> A549> CCC-HEH-2> SH-SY5Y（basedon IC10values);7) busulfan: HEK-293> HepG-2> CCC-HEH-2> SH-SY5Y> A549(based on IC50and IC10values);8) cadmium chloride: HEK-293> HepG-2>SH-SY5Y> CCC-HEH-2> A549(based on IC50values), HEK-293> CCC-HEH-2>HepG-2> SH-SY5Y> A549(based on IC10values). The toxicity effects to different cellsare listed below (exposed for72hours):1) paraquat: HEK-293> HepG-2> A549>SH-SY5Y> CCC-HEH-2(based on IC50and IC10values);2) busulfan: HEK-293>HepG-2> SH-SY5Y> A549> CCC-HEH-2(based on IC50and IC10values).High targeted properties of these cell lines were detected based on the IC50and/or IC10values with the exception of A549to paraquat and busulfan which are positive toxicants tolung in vivo. In addition, A549had the lowest IC10in co-culture to cyclophosphamide(results showed in the section below), predicted lung is the target-organ correctly.3. To detect the metabolic abilities of hepatic cell lines in the IdMOC systemTo detect the metabolic abilities of HepG-2by comparing the toxicity effects to theother4cell lines of acetaminophen and cyclophosphamide which exhibit toxic effects bytheir metabolites, and using invtro micronucleus test with cyclophosphamide in HepG-2.In the IdMOC system, the acetaminophen and cyclophosphamide showed higher toxicityeffects on A549，HEK-293，SH-SY5Y and CCC-HEH-2than those cultured separately.Micronucleus rates increased in HepG-2traeated with cyclophosphamide. Indicated,HepG-2has some metabolic activity on acetaminophen and cyclophosphamide.4. Primary study in using of the IdMOC system to evaluate the toxicity effectsof3nanometer materialsThree kinds of nanometer materials of zinc oxide (ZnO,30nm,50nm,100nm, andmico-scale), Silicon dioxide（SiO2,15nm,30nm,50nm,100nm, and mico-scale）andcadmium sulfide quantum Dots (CdS quantum dots; fluorescence λem480nm (size1.6nm),420nm(size between1.6nm and7.3nm),480nm(size7.3nm), and mico-scale） were evaluated in the IdMOC system using MTT method. The primary target-cell fornano-ZnO is HEK-293, and they also showed remarkable toxic effects on SH-SY5Y andHepG-2as the nano-ZnO size became smaller. Indicated, kidney, neural system and liverwere possible target-organs for nano-ZnO in smaller size too. The primary target-cell fornano-SiO2is SH-SY5Y, and they also showed remarkable toxic effects on HepG-2andHEK-293as the nano-SiO2size became smaller. Indicated, neural system, kidney andliver were possible target-organs for nano-ZnO in smaller size too. The most sensitivecell line is HEK-293to CdS quantum dots, followed by HepG-2and SH-SY5Y. Indicated,kidney is the primary target-organ to CdS quantum dots, followed by liver and neuralsystem.In general, the toxicities of these3types of nanometer materials are higher than theywere in micro scale, respectively. With the size became smaller of ZnO and SiO2, toxicityeffects became stronger, more target-organs were obtained. CdS quantum dots withfluorescence λem420nm showed the highest toxicity effects compared with the other2kinds of CdS quantum dots, and their target cell is HEK-293.Conclusions:1) It was the first time to establish the system of integrated discrete multiple organco-culture using human originated cell lines (IdMOC).2) The IdMOC model of human originate cells can be used as research model fortoxicity target-organ screening by primary validation. Target-organs of toxicantscan be recognized by comparing values of their IC50and/or IC10values on differentcells. Target-organs of toxicants in low exposure can be found out more effectivelywhen combining the values of IC50and IC10to be evaluated. This study is verypreliminary, only limited toxicants were tested. The cell lines used and toxicityindexes evaluated still need to be further optimized and validated.3) Cell HepG-2inherited some metabolic abilities from liver, and can active theacetaminophen and cyclophosphamide to show higher toxicity effects.The toxicities of these3kinds of nanometer materials (ZnO, SiO2, and CdS quantumdots) are higher than they were in micro scale, respectively. Target-organs for bothnano-ZnO and CdS quantum dots might be kidney. More target-organs, and highertoxic effects were observed in this system when the size became smaller of nano-ZnO and nano-SiO2. The IdMOC model of human originated cells may be used as a newmethod in toxicity evaluation of nanomaterials．...