Study On Toxic Effect Of CML Of Caenorhabditis Elegans

Backgrounds Carboxymethyl lysine(CML),a marker of glycation endproducts(AGEs)with clear structure which is liable to be produced in the Maillard reaction in food processing,is potentially harmful substance widely exist in food.As a new harmful substance in food,there are a few reports on CML,and scholars have not formed a conclusion on it.As a typical biological model,Caenorhabditis elegans are widely applied in scientific research fields.Because they are easy to preserve,culture,and has well-developed synchronization technology,stable genes for operation,short diverse life cycle,tiny size and transparent surface for observation.This study intends to explore the toxicity effects of CML on Caenorhabditis elegans by researching its lifespan,the number of offsprings and the survival time in thermal environment when exposed to different conventration CML.Methods1.Detect the effect of different concentration CML on the lifespan of Caenorhabditis elegans;2.Intervene the Caenorhabditis elegans with different concentration CML from the zygote period and research its influence on the number of offsprings;3.The survival time of Caenorhabditis elegans was observed with thermal stimulation under different concentrations of CML;4.The mRNA expression of genes related to reproduction and lifespan of Caenorhabditis elegans was detected by fluorescence quantitative PCR,and the possible mechanism of toxicity of CML was explored.Results 1.CML had toxicity effect on the lifespan of Caenorhabditis elegans and showed a dose-response relationship.The greatest effect was in 50mmol/L group,and the average life expectancy of Caenorhabditis elegans reduced by 123%.2.CML had toxicity effect on nematode reproduction of Caenorhabditis elegans: CML shorten nematodes spawning period,and discharge nematode fertilized eggs shortly;there is no significant change in the number of Caenorhabditis elegans offspring in 0.08mmol/L,0.4mmol/L and 2mmol/L CML group,but significant decrease in 10mmol/L and 50mmol/L CML group.3.CML had effect on the survival of Caenorhabditis elegans in thermal environmental: survival curves of Caenorhabditis elegans moved left under CML treatment.In 0.4mmol/L,2mmol/L CML group,the survival curve did not significantly change compared to control group before 12 h,but there are significant differences in the intervention group after 12 h.In 0.08mmol/L,10mmol/L,50mmol/L group,the significant differences were also detected.The results suggested that CML affected on the survival of Caenorhabditis elegans in a dose-response relationship.4.The mRNA expression levels of Caenorhabditis elegans lifespan and reproductive-related genes showed change in different degrees when exposed to CML.Daf-16 had no significant change in the low concentration of CML(0.08 mmol / L,0.4 mmol / L,2 mmol / L,but showed a significant decrease(P10mmol/L<0.05、P50mmol/L<0.05)in the high concentration of CML(10mmol / L,50 mmol / L).The mRNA expression levels of SKN-1,Hsf-1 significantly increased when exposed to 0.4mmol/ L CML(Phsf-1 0.4mmol/L<0.05,PSKN-1 0.4mmol/L<0.05).The mRNA expression levels of Folt-1 significantly increased when exposed to CML(P0.08mmol/L<0.05,P0.4mmol/L<0.01,P2mmol/L<0.01,P10mmol/L<0.01,P50mmol/L<0.01).There was no significant change in the mRNA expression of Daf-2,JNK-1,daf-12,pha-4,fer-1,spe-10,spe-15 under the CML treatment.Conclusions CML as a free state of low toxicity substance,affected Caenorhabditis elegans’ lifespan,reproduction,and survival time under thermal environment.Because of its deposition in the body,the toxicity of CML on Caenorhabditis elegans gradually increased with the increase of exposure time.And the effect of CML on Caenorhabditis elegans lifespan,reproduction,survival time under thermal environment showed a dose-response relationship.Through skn-1 of the TOR signaling pathway and hsf-1 of the reproductive system signaling pathway,CML enhanced Caenorhabditis elegan’ toxicity cleaning function and resistance against thermal environment and thus prolonging its life.But DAF-16 expression decreased significantly in high concentration,which indicated that high dose of CML significantly influenced the life activity and lifespan of Caenorhabditis elegans.This was proved by the expression of folt-1: under the low-dose treatment of CML,folt-1 expression was significantly increased,but the number of offsprings did not change;whlie in high-dose of CML treatment,the offspring numbers of Caenorhabditis elegans reduced significantly.