Seeking For Highthrough-put Methods To Detect The In Vitro Mutagenicity Of Chinese Traditional Medicines

Traditional Chinese Medicines (TCMs) are important resources to resolve the problem of bacterial resistance during the post antibiotics era. TCMs are the main contents of Chinese medium and long-term science and technology development program. At the same time, TCMs are more and more accepted by the people all over the world. Therefore, the safety of TCMs is paid more and more attentions.TCMs are always used as the decoctions of many kinds of TCMs directed by the doctors. So we have to know the safety of these TCMs individually before understanding the safety of TCMs compounds. Although there is a long history of the usage of TCMs, there is still not a database about the safety of TCMs. Therefore, the porpuse of this study is to modify the previous methods and construct new methods to suit the safety evaluation of TCMs. These methods are all based on microorganisiums and used to screen the genotoxicity of TCMs. We are trying to develop a database about the safety of TCMs and there cmpounds which are used often and offer reasonable references for the clinical usage of TCMs.There are four procedures in drug safety evaluation progress including acute toxicity test, genotoxicity test (including mutagenicity), subchronic test and chronic toxicity test. Only the genotoxicity test can be implemented with the microbial technology among these tests. A large number of data indicates that there is a certain correlation between the mutagenicity and carcinogenicity of samples, so the carcinogenicity of Traditional Chinese Medicines could be predicted by their mutagenicities.According the the special characteristics of TCMs, the jobs that we have done in this study are as follows:1. There are many in vitro mutagenicity testing systems and classical bacterial reverse mutation test (Ames test) is most widely used. There are some disadvantages for the Ames test in detecting in vitro mutagenicity of Traditional Chinese Medicines. For example, the results of Ames test will be influenced by the histidine in samples. The clasical Ames test was modified in this study to satisfy in vitro mutagenicity detection of Traditional Chinese Medicines. Over-dose histidine was added into the detection systems and the influorences of traces of histidine in samples were coveraged. After this modification, it could be used to detection the decotion of TCMs. A fixed treat time is used in State Standards while different samples may need different time to be treated. A suitable time needs to be found to make sure that all the mutagenicities of samples could be detected. Results suggested that the modified method needs more modifications to evalution the safety of TCMs. Ames test is too complicated to be suitable for rapid evaluation of a large number of Traditional Chinese Medicines. The aim of this subject is to find a high-throughput method to replace Ames test for the mutagenicity detection of Traditioal Chinese Medicines. Results of the modified Ames test can be used as the reference of other methods, after all, there are standards for the Ames test.2. Since the1980s, more and more substitutes of the classical Ames test have emerged. One of them is umu-lacZ test which is based on SOS response and the results of this system are well consistent with Aems test. SOS response will be promoted when the cell is damaged by mutagens and the umuDC genes will be expressed more proficiently. We can detect the mugagenic charactaristics of different samples by determine the enzyme activity of (3-galactosidase which is the gene product of lacZ gene. At present, this method is widely used for mutagenicity testing of drinking water and sewage, and international standard has been developed for this method. To avoid the detection of enzyme activity, we construct the umu-EGFP system. The sensitivity of the umu-EGFP system is too low to be used in mutagenicity detection of Traditional Chinese Medicines.3. To enhance the sensitivities of our test system, we substituted the umuDC gene promoter for the recA gene promoter and constrcted the rec-EGFP system. This system could be used in detecting the mutagenicity of samples after verified by positive mutagens. Compared with the umu-lacZ system and animal experiments, the rec-EGFP system has a higher positive rate. Although we have not known the reason for this phenomenon, the positive results can’t be neglected because this system could detect positive mutagens well. More research needs to be done to explore the reasons for the high positive results of rec-EGFP system when it is used to detect genotoxicity of TCMs.4. Finally, we have tried to modify the micro-well plate method of umu-lacZ system to conquer the interference of Traditional Chinese Medicines’ colors by centrifuging and resuspending the cells with normal saline solution. Finally, the modified umu-lacZ system can be used to detect the mutagenicity of Traditional Chinese Medicines in a high-throughput way and the results of umu-lacZ test are consistent well with previouis chromosome aberrations test.5. We have detected dozens of Traditional Chinese Medicines with the umu-lacZ test. We will detection more Traditional Chinese Medicines with modified umu-lacZ system and establish a database to offer references for reasonabale clinical medication.The results of this study will offer references for the modification of State Standards for Drug Safety Evaluation Progress and pave the way for constructing new standards.