| The Niuhuang jiedu tablet is a representative of Qingrejiedu prescription in TCM, usually using for filled hot, sore throat, sore gums, tongue festered, red and swelling eyes and etc. For the evaluation Niuhuangjiedu security, the Ames test, chromosome aberration analysis, single cell gel electrophoresis assay (SCGE) and micronucleus tests were used, respectively, to explore the potential genotoxic effects of Niuhuang jiedu tablet. In addition, compatibility detoxification is one of the most characteristic methods and techniques for TCM, to explore whether the compatibility for Niuhuang jiedu tablet could reduce the toxicity of realgar and what is its possible mechanisms, different groups about Niuhuangjiedu tablet and its disassembled presciption were arranged, and soluble arsenic, arsenic in the body tissue distribution, pathological damage, oxidative damage, genetic damage, apoptosis etal, were detected.1Genetoxicity evaluation for Niuhuang jiedu tablet1.1Genetoxicity studies of Niuhuang jiedu tablet in in vitro bioassays1.1.1Effects of Niuhuangjiedu tablet on gene reverse mutation Plate incorporation assay was adopted, in accordance with Maron, DM and Ames BN recommended procedures. Based on the pre-test results,5mg/dish was arranged as the highest dose group, then,1.5,0.5,0.15,0.05mg/dish, negtive and positive control groups were also arranged to carry out the experiment. The results showed no difference on gene reverse mutation of Trphoid Salmonella Strains, from0.05mg/dish to5mg/dish, both with and without S9mixture, and replies colonies number of various strains did not exceed2times the negative control group, and dose-response relationship was not showed.1.1.2Chromosomal aberrations effect of Niuhuangjiedu tablet on CHL cells The IC50of Niuhuangjiedu tablet was determined as54μg.ml-1, by cell counting method. Dose range for chromosome aberration test was1.0,1/2,and1/4IC50values, i.e,54,27.13.5μg.ml-1respectively. The results showed that chromosome aberration rate of all dose group were less than5%, at24hrs exposure, and at9hrs exposure with the activation system S9, which is a negative response, chromosome aberration rate of the middle dose group was8.5%, which is greater than5%, and is a suspicious positive, at48hrs exposure, but chromosome aberration rate of high and low dose group is less than5%.1.1.3DNA damage effect of Niuhuangjiedu tablet on CHL cells The DNA strand break effect of Niuhuangjiedu tablet on CHL cells was tested by SCGE. Dose range was also1.0,1/2,and1/4IC50values, i.e,54,27.13.5μg.ml-1respectively. The results showed DNA damage rised at the54μg.ml-1dose group after48hrs exposure in comparation with the negative control group, and the difference was significant (P<0.05), but there were no difference was found at medium and low dose group, which means Niuhuangjiedu tablet could induce DNA strand break of CHL cells at high dose.1.2Genetoxicity studies of the Niuhuangjiedu tablet in in vivo assays1.2.1The influence of Niuhuangjiedu tablet on chromosome integrity and separation changes Bone marrow PCE cell micronucleus test in mice exposed Niuhuangjiedu tablet through oral delivery with three dose groups (3345,1670,835mg.kg-1) was carried out, and two administering drugs regimens ("0" and "24" hrs administration at twice and6weeks continuous administration) were arranged. The results showed that the "0" and "24" hrs administration at twice, no significant difference (P>0.05)was found at each dose group,which suggested that under this regimen, the Niuhuangjiedu tablet had no influence on mice chromosome integrity and chromosome segregation. But under6weeks administration regimen, micronucleus rate at dose of1672mg.kg-1and3345mg.kg-1was found higher than that of negative control group (P <0.05), and the obvious dose-response relationship (r=0.999, P=0.023)was found, which means Niuhuangjiedu tablet could increase the mouse bone marrow cells micronucleus rate under long time administration.1.2.2Sperm abnormality test in mice exposed to Niuhuangjiedu tablet Sperm abnormality test in mice exposed to Niuhuangjiedu tablet was carried out with three dose groups (3345,1670,835mg.kg-1), and the two administering drugs regimens (5d days administration and6weeks administration) were arranged. The results showed each dose group and the negative control group showed no significant difference (P>0.05) under two regimens, which means Niuhuangjiedu tablet has no significant genetic damage effect on germ cells in mice under this experimental conditions.1.2.3DNA strand break of main tissues and organs cells in mice exposed to Niuhuangjiedu tablet SCGE technique was used to detect DNA strand breaks of peripheral blood cell in mice exposed to Niuhuangjiedu tablet, at different dose (3345,1670,835mg.kg-1) for different administration times (1w,2w,3w,4w and5w), and after the last administration, DNA strand breaks of mice lung, liver, kidney, spleen cell were also detected. The results showed that the DNA of peripheral blood lymphocytes began to perform strand breaks at administration2w or3w with high dose, and then significant DNA strand breaks were detected at all dose groups after administration4w or5w. Significant DNA strand breaks were also detected in cells of liver, lung cells at high and medium dose groups, and in renal cell at high dose groups, when compared with negtive control group, which indicates that Niuhuangjiedu tablet may induce DNA damage of liver, lung and renal cell after administration6w and through oral delivery.Preliminary Conclusion:Niuhuangjiedu tablet had no obvious genetoxicity in in vitro and vivo experimental conditions, but with the increase of administration dose and the extension of administration time, which may have cumulative genetoxicity. 2The preniminary exploration on Niuhuangjiedu compatibility reducing realgar arsenic toxicity2.1Determination of Soluble Arsenic in NiuhuangJiedu Tablet and its disassembled presciption Different groups about NiuhuangJiedu Tablet and its disassembled presciption were arranged, treated with artificial gastric juice or artificial intestine juice, then Atom Fluorescence Spectrophotometry(AFS) was used to detected the content of soluble Arsenic in the different groups samples. The result showed the content of soluble Arsenic in the whole presciption of NiuhuangJiedu Tablet was significant lower than that in the single relgar (P<0.01), the content of soluble Arsenic in the whole presciption with Gancao,Hangqin,Dahuang excluded was significant higher than that in the whole presciption of NiuhuangJiedu Tablet (P<0.01),which suggested the compatibility for Niuhuangjiedu Tablet could reduce the toxicity of realgar,and Gancao,Hangqin and Dahuang may be the possible herbal medicines that play the role of reducing toxicity.2.2Animal experiment studies on Niuhuangjiedu Tablet compatibility reducing realgar arsenic toxicity2.2.1The influence of Niuhuangjiedu tablet compatibility on the absorption and distribution of arsenic in realgar The AFS was used to detect and analye the influence of Niuhuangjiedu tablet on the absorption and distribution of arsenic in realgar. The results showed arsenic content in liver, kidney, spleen, heart and lung tissue in the single realgar was significantly higher than that of negtive group after administration9W (P<0.05), which confirmed that the arsenic in realgar could be absorpted and distributed in bodies. Compared with the whole presciption of NiuhuangJiedu Tablet, arsenic content in liver, kidney, spleen, heart and lung tissue in realgar was significantly higher (P<0.05), and arsenic content in liver, spleen, heart tissue in the whole presciption with Gancao,Hangqin,Dahuang excluded was also significant higher (P<0.05) which suggested the compatibility for Niuhuangjiedu Tablet could reduce the absorption and distribution of arsenic in realgar, and the absorption and distribution of arsenic in realgar may increase if Gancao, Hangqin.Dahuang in whole presciption of NiuhuangJiedu Tablet are excluded.2.2.2The influence of Niuhuangjiedu tablet compatibility on the oxidative stress The lipid peroxide product MDA and antioxidant enzymes SOD and GSH-px were determined to explore the influence of Niuhuangjiedu tablet compatibility on the oxidative damage. The results showed GSH-px enzyme activity in the liver, kidney, lung, heart tissue and SOD enzyme activity in liver kidney, spleen tissue decreased significantly (P<0.05).and MDA content in liver, kidney, heart tissue increased significantly (P<0.05),when single realgar compared with the negtive group. However, the GSH-px and SOD enzyme activity and MDA content in the whole presciption and in the whole presciption with Gancao, Hangqin, Dahuang excluded showed no significant change (P>0.05), which indicates that the realgar could cause oxidative damage, and Niuhuangjiedu compatibility could reduce oxidative damage caused by realgar, but which are the main herbs that play the role of antioxidative damage may not be determined.2.2.3The influence of Niuhuangjiedu tablet compatibility on genetic damage Comet technology, the micronucleus test, etc were used to determined the influence of Niuhuangjiedu tablet compatibility on genetic damage. The results showed the DNA damage in peripheral blood cell, micronucle formation rate in bone marrow PCE cell, and the sperm abnormality rate in single realgar increased signiffcantly (P<0.05),when compared with the negative group. DNA damage, micronucleus formation rate decreased signiffcantly (P<0.05), sperm abnormality rate had no signiffcant difference in the whole presciptio of Niuhuangjiedu tablet in comparation with single realgar; DNA damage, micronucle formation rate and sperm abnormality rate in the whole presciption with Gancao,Hangqin,Dahuang excluded showed no significant change. These results indicates that the Niuhuangjiedu compatibility could reduce the genetic injury of realgar, but the specific herbs that play role of antigenetic damage was not yet determined.2.2.4The influence of Niuhuangjiedu tablet compatibility on liver and kidney toxicity The pathological and hisological observation was used to analye the influence of Niuhuangjiedu tablet compatibility on liver and kidney toxicity of realgar. The results showed that liver tissue in the realgar was significantly edema, local necrosis; and local renal tubular cells in kidney tissues in the realgar was severe edema; The liver and kidney tissues showed no abnormal changes except local edema in the whole presciption of NiuhuangJiedu Tablet and the whole presciption with realgar excluded, which suggested Niuhuangjiedu tablet compatibility may could reduce the liver and kidney toxicity of realgar. The liver tissue showed mild edema and kidney tubular epithelial cells showed moderate edema in the whole presciption with Gancao, Hangqin, Dahuang excluded, which indicates the liver and kidney toxicity may be enhanced with Gancao, Hangqin, Dahuang removed from the whole presciption of NiuhuangJiedu.2.2.5The influence of Niuhuangjiedu tablet compatibility on liver apoptosis The TUNEL and immunohistochemical methods were used to determine the influence of Niuhuangjiedu tablet compatibility on liver apoptosis and apoptosis related molecules expression. Results showed the TUNEL positive cell numbers and mean density of Bax expression increased significantly, but mean density of Bcl2expression reduced significantly, when realgar group compared with negative group (P<0.05). However, TUNEL-positive cell numbers and mean density of Bax expression decreased significantly, and mean density of Bcl2expression increased significantly, when the whole presciption compared with the single realgar (P<0.05), indicates the Niuhuangjiedu compatibility may inhibit liver apoptosis that realgar induced, and such inhibition may be achieved by promoting bcl2expression and inhibiting bax expression. The whole presciption with Gancao, Hangqin, Dahuang excluded in compared with the whole presciption, the TUNEL positive cell numbers and mean density of Bax expression increased significantly, but mean density of Bcl2expression had no apparent changes, which indicated Gancao, Hangqin. Dahuang in the whole presciption may reduce the liver toxicity of realgar by antiapoptosis.Preliminary Conclusion:The compatibility of Niuhuangjiedu tablet may reduce the asenic toxicity of realgar, and reducing absorption and distribution of soluble arsenic, antioxidative damage, antiapoptosis, and et al maybe the possible mechanisms. The specific herbs that reduced asenic toxicity of realgar were not yet determined in this study, which needs further research.
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