An Evaluation Of The Genotoxicity Of F.cirrhosa And The Underlying Molecular Mechanisms

Bulbus of Fritillaria cirrhosa(BFC)is a renowned traditional medicine and has been officially recorded in the Pharmacopoeia of the People's Republic of China.BFC has a long history of being used as a folk midicine and has remarkable ability to treat various lung symptoms and diseases,specifically,cough and asthma.The genotoxicity of drugs refers to their potential in disrupting the integrity of DNA and chromosomes,either directly or indirectly.DNA damage and chromosomal instability(CIN)give one of the biggest risks for human health,since CIN is an ultimate linker to organ/tissue aging and aging-related chronic diseases,such as cancer,neuro-degenerative diseases,cardiovascular disease and many others.However,little attention is being paid to determing whether BFC has genotoxicity.To address these questions,the normal human colon mucosal epithelial cell line,NCM460,was chosed as an in vitro cell model.Moreover,several methods,including(?)cytokinesis-blocked micronucleus assay to determine CIN by using the biomarkers of micronucleus(MN),nucleoplasmic bridge(NPB)and NB(nuclear bud);(?)the mitotic aberrations assay to determine the mitotic errors by using the biomarkers of chromosome misalignment(CMA),multipolar alignment(MPA),laging chromosomes(LC),chromatin bridge(CB)and multipolar segregation(MPS);(?)the nocodazole-chanllenge assay to determine the function integrity of SAC(a surveillance mechanism against CIN);(?)the cell synchronization by using thymidine,nocodazole and cytochalasin B;and(?)the RT-qPCR to determine the transcriptal expression of genes,were selected in the present study.By using these materials and methods,we investigated the genotoxicity of BFC and its bioactive components peimisine and imperialine,and the potential molecular mechanisms underlying them.Our results showed that(?)under short-term treatment,BFC at 80-160 ?g/mL could significantly induce MN and NB.The molecular mechanisms underlying BFC-induced MN probably involves aneugenic,but not clastogenic processes.Under long-term treatment,20-40 ?g/mL BFC could also significantly induce MN and NB.In addition,the CIN induced by BFC is long-lasting.(?)BFC at 80-16?g/mL could significantly arrest cells in mitosis,which was associated with the fact that BFC induced CMA in metaphases.BFC could interefere the mitosis progression and arrest the mitotic cells in prometaphase.In addition,BFC could weaken the activity and function of SAC,leading to the generation of LC and CB during ana-telophases.The molecular mechanisms underlying BFC-induced SAC weakened is the dysregulation of the expression of several SAC genes,such as the down-regulation of BubRl,Mad2,CENP-E and Aurora-B and the up-regulation of Aurora-A,Plkl9 Mps1,Bubl and Madl.(?)BFC at 80-160?g/mL could significantly induce mutlinucleation by inducing cytokinesis failure(CKF).The CKF induced by BFC might result from the defects in forrow ingression of the contractile ring,since BFC has no effect on the expression of RhoA and Anillin,two center genes involved in contractile ring assembly,but it significantly inhibited the expression of LATS1 and LAST2,two master genes involved in furrow ingression.(?)80-160 ?g/mL could significantly induce mitotic spindle multipolarity.The mitotic multipolarity is not due to the promotion of binucleated cells into mitosis,but the premature separation of centrosomes in mitosis,which resulted in centrosome over-amplification.In addition,BFC could inhibit the expression of genes involved in preventing centrosome over-amplification,such as Rb,p53,Plk4 and Centrin-2,and increase the expression of YB-1,a gene promotes centrosome amplification.(?)Verticine,verticinone,imperialine and peimisine could significanlty induce MN,NPB and NB under the concentrations that were not cyotoxicity(5-80 ?g/mL),in a time-and dose-dependent manner.In addition,imperialine and peimisine has a synergistic effect in promoting MN and NB at a concentration that had no single-agent genotoxic effect(2 p.g/mL),although this synergistic effect was weakened as time increased.In summary,this study demonstrated that BFC at high doses(80-160 ?g/mL)possessed genotoxicity.Our results further supported the genotoxicity of BFC could be resulted from(?)the compromised SAC and the subsequent aberrant chromosome alignment and segregation;(2)cytokinesis failure;and(3)centrosome premature separation and amplification,these aberrant events would ultimately destory the ingerity of genome.In addition,the bioactive alkaloids verticine,verticione,imperialine and peimisine were the potential genotoxins in BFC,and imperialine and peimisine had synergistic genotoxic effects.