Investigation On The Cytotoxicity Mechanism Of Quinocetone In Vitro

Quinoxaline-1,4-dioxide, as veterinary drugs, is a kind of non-hormonal animal feeds widelyconsumed in the world. Quinocetone, as one number of the quinoxaline-1,4-dioxide, is a new veterinarydrug in China, and is an international initiative veterinary drug. Since previous studies havedemonstrated that the other numbers of quinoxaline-1,4-dioxide, carbadox and olaquindox are mutagens,and since quinocetone has the same parent nucleus structure as that of carbadox and olaquindox, the useof quinocetone in animal feed has raised concerns. The studies from the chemical structure factors,oxidative damage, genotoxicity and gene level were to analyze the cytotoxicity mechanism ofquinocetone in vitro. The results gained will provide a basis for rational evaluation of the safety ofquinocetone.In this study, we successfully gained27compounds with structure similar to quinocetone andbisdesoxyquinoceton. Quinocetone and its structure similar compounds had antibacterial activity.Generally, their antibacterial activities were very close. The compounds from quinocetone branchedhydroxyl modification was slightly better than quinocetone, however, for the compounds fromquinocetone parent nucleus deoxidization, the antibacterial activity rapidly disappear. Quinocetone andits structure similar compounds had obvious effect on the HepG2, Chang Liver and Vero cells growthinhibition, and their inhibition effects were far higher than that of olaquindox, MQCD and otherstructure similar compounds. The results also showed that the cell growth inhibitions of the compoundsfrom quinocetone parent nucleus deoxidization were significantly decreased. These results indicatedthat quinocetone mother nucleus N→O groups was required for its biological activity, and double bondand carbonyl in branched chain had significant influence on the cell toxicity of quinocetone. we furtherused the LC/MS/MS technology to study the metabolism of quinocetone and other compounds onHepG2cells in vitro. The results showed the parent nucleus deoxidation and the reduction of doublebond and carbonyl in branched chain was their primary metabolism way. The results further proved thatthe parent nucleus deoxidation was related to their cytotoxicity.The study found that quinocetone could significantly increase malondialdehyde concentration,damage many anti-oxidation systems, and significantly reduce the cellular antioxidant capacity inHepG2cells. When the cells were co-treated with quinocetone and glutathione synthesis inhibitorDL-buthionine sulfoximine (BSO), the cell growth inhibition rates of quinocetone were significantlyincreased. however, the cells were co-treated with quinocetone and glutathione synthesis promoterN-acetylcysteine (NAC), the cell growth inhibition rates changed a little. The results suggested thatoxidative damage was quinocetone cytotoxic important way. In addition, quinocetone couldsignificantly induced micronuclei in HepG2cells, and the nuclear division index (NDI) decreased, andquinocetone could increase DNA random amplified polymorphism, induce HepG2cells apoptosis,change the cell cycles and induce the S phase arrest. We built differentially expressed cDNA library of the HepG2cells after exposure to quinocetoneby bidirectional subtractive hybridization. By sequencing and BLAST alignment analysis, more than160different expression genes were gained, including96forward differentially expressed genes and68reverse differentially expressed genes. The function mainly focused on drug metabolism, drug delivery,oxidative stress, translation regulation, protein synthesis, cell growth, cell cycle and apoptosis. Theresults indicated that biological effects affected by quinocetone had diversity. The real-time PCR resultswere consistent with that of the subtractive hybridization and the changes of ENO1, AKR1C1, DDX5,AIFM and TNFRSF gene expression suggested the myc signal transduction pathway or mitochondriamaybe mediate the apoptosis induced by quinocetone.In a word, the cytotoxicity mechanism of quinocetone may be (1) on chemical structure,branched-chain carbonyl group, a double bond and a mother nucleus N→O group together make up thetoxicity group, and increase its cytotoxicity;(2) on action mode, oxygen free radicals deoxy fromquinocetone nucleus damage proteins, DNA and other biological macromolecules, resulting in increasedmicronuclei, DNA amplified polymorphic and other toxic effects;(3) on molecular mechanism, ENO1,AKR1C1and other enzymes may be involved in the metabolism of quinocetone in cells, and furtherstimulate the myc signal transduction pathway or mitochondria pathway, leading to the genetic materialinjury and cell apoptosis.