The Toxicity And Metabolism Research Of 3-Bromopyruvate On Caenorhabditis Elegans

Objective: To observe the toxic effect, larva development time, fat content and the related genes expression of Caenorhabdities elegans(C. elegans) after the treatment of 3-bromopyruvate. RNA interference and mutant strains were used to explore the toxic effect and the metabolic mechanism of 3-bromopyruvate.Methods: C. elegans was employed as the model organism to be treated with 3-bromopyruvate on the NGM plate, the status of C. elegans and the numbers of survivors were observed every 24 hours under the optical microscope. RNA isolation was proceed after C. elegans were treated with 3BP for 24 hours. The expression of genes related to toxicty the drug metabolism was measured by Real-time quantitative PCR. Three hexokinase genes were chose for RNA interference, mutant strains and RNAi feeding strains were used to explore the mechanism of 3-Bromopyruvate.Results: 1. The development time of larva cultivated on NGM which was mixed with 3-bromopyruvate was significantly prolonged(P<0.05).2. Record the survivors after C. elegans were tainted with 3-bromopyruvate for 24 hours and the LC50 was 12.7mmol/L that was calculated by SPSS 19.0.3. Apoptosis was not discovered in somatopiasm and germ cells after C. elegans were tainted with 3-bromopyruvate for 24 hours.4. The fat in head and intestine was increased after C. elegans were treated with 3-bromopyruvate for 72 hours.5. The Real-time quantitative PCR results showed that all the hexokinase genes named hxk-1, hxk-2 and hxk-3 were overexpressed after the treatment of 3-bromopyruvate.6. We had successfully interfered three genes(hxk-1, hxk-2 and hxk3) of C. elegans with RNAi method. All the LC50 of RNAi C. elegans were decreased.7. Hexokinase Phylogenetic Comparison was processed with Clustal X and MEGA5.05. hxk-1 and Necator americanus belonged to a branch with a higher similarity. hxk-2 and hxk-3 of C. elegans belonged to a branch with a higher similarity, but had rather distant phylogenetic relationships with hxk-1.8. All the CYP35 were overexpressed excepct cyp-35B3, and cyp-35A1 was the most obvious one with overexpression.9. The LC50 of cyp-35A1 mutant, cyp-35A2 mutant, cyp-35A4 RNAi C. elegans and cyp-35B3 RNAi C. elegans were lower.Conclusion: The development time of larva was prolonged obviously after the treatment of 3-bromopyruvate in a concentration-dependent manner. All the hexokinase genes were up-regulated to different extent. The phylogenetic tree indicated that hxk-2 and hxk-3 were closer than hxk-1. The gene cyp-35A1 was the most obvious up-regulated one, which mainly involved in the metabolism of 3-bromopyruvate, the down-regulated expression of cyp-35B3 gene showed that 3-bromopyruvate may inhibit its expression, or the up-regulated expression of cyp-35A1 had an effect on the expression on cyp-35B3.