Study On Cytotoxicity And Metabolomics In Cells Treated With The Combination Of Methamphetamine And Ketamine

Psychotropic drugs are a kind of drugs that have excitatory or inhibitory effects on the central nervous system.In recent years,the problem of drug abuse is still severe on a global scale,and the pattern of drug abuse is gradually turning to polydrug abuse,which has brought more negative effects on the physical and mental health of abusers.Therefore,the harm of drug abuse to the body is also receiving increasing attention.Methamphetamine(MA)and ketamine(KET)are two of the most widely abused psychoactive drugs,and most domestic and foreign studies on the toxicity of these two substances are conducted under the action of a single drug,ignoring the combined toxic effects of two or more drugs in the case of multi-drug abuse.In order to study the cytotoxicity of MA,KET and their binary mixtures,this paper analyzed the combined effects of MA and KET on oxidative stress,apoptosis,genotoxicity and metabolome interference by factorial design and variance analysis,and preliminarily discussed the mechanism of the interaction between MA and KET on Hep G2 cells.Both MA and KET were found significant cytotoxicity indicated by the viability of Hep G2 cells,and MA showed stronger cytotoxic effect.The mixtures of MA+KET showed higher cytotoxicity than single MA or KET treatments.Additive effect was found in the 0.1+0.1 group,and synergistic effects were found with the increasing concentration of MA or KET.Oxidative damage effect of target chemicals and its mixture on Hep G2 cells was evaluated by the determination of MDA content,SOD activity,CAT activity and GSH content.In single treatments,both MA and KET induced oxidative stress,and MA showed stronger effect of oxidative damage.MA+KET combined treatments showed stronger oxidative damage effect compared with single treatments.For different research indexes,different concentration combinations have different types of combined effects,For the combined groups,additive effect was foun in the combination of low concentration drugs,but the interaction type changed from additive effect to synergistic effect when the concentration of MA or KET increased to 1.0 m M.The possible reason for the combined effect was the enhancement of KET metabolism by MA.Apoptosis rate,mitochondrial membrane potential,Caspase-3 activity and adenosine content in Hep G2 cells were determined to investigate the apoptotic effect of the target drugs and their mixtures on Hep G2 cells.Both MA and KET induced apoptosis and mitochondrial damage in single treatments,and higher mitochondrial toxic was found when the cells were treated with KET.For the combined effect of MA+KET,the apoptotic rate and mitochondrial damage were higher than those under the single effect,and all the combined groups showed synergistic effects except the low concentration combined group.The enhanced apoptotic effect of MA+KET mixtures was possibly related to effect that KET promoted the migration of pro-apoptotic protein to mitochondria.Comet assay and cell cycle assay were used to investigate the DNA damage effects of the target drugs and its mixture on Hep G2 cells.Significant DNA damage and G2/M arrest were observed in Hep G2 cells after exposed to MA and KET,indicating that both drugs caused genotoxicity and the DNA repair disorders may be involved in the mechanism of their genotoxicity.Compared with the single treatment groups,the mixtures of MA+KET aggravated the DNA damage and G2/M arrest in Hep G2 cells,indicating that more severe genotoxic effects were occurred.The results of three indexes(tail DNA percentage,tail length and Olive tail moment)in comet assay indicated the combination of low concentration of MA and KET showed additive effect on DNA damage.But the type of interaction turned into synergistic effect when the concentration of MA or KET increased to 1.0 m M.The possible reason for the combined effect was that MA could inhibit the repair of DNA.Untargeted LC-MS metabolomics was used to investigate the effect of the target substance and its mixture on metabolome in Hep G2 cells.In the single treatment of MA or KET,glutathione metabolism,alanine-aspartate-glutamate metabolism and sphingolipid metabolism were the most disturbed metabolic pathways in Hep G2 cells.In the treatments of MA+KET,the disturbances of some metabolic pathways in single effect were enhanced,suggesting that MA and KET may produce combined toxicity through the disturbance of energy metabolism and purine metabolism in Hep G2 cells.Cytotoxicity test,oxidative damage assays,apoptotic assays,genotoxicity assays and metabolomic analysis show that the MA and KET caused obvious effects of apoptosis,oxidative damage and genotoxicity in Hep G2 cells.Moreover,the energy metabolism,lipid metabolism and oxidative stress related metabolism were disturbed after drug treatments.The combined toxicity of MA+KET mixtures were more severe,and the type of combined effects were additive or synergistic interaction.The results of this study provide a basis for evaluating the cytotoxicity and combined toxicity of psychotropic drugs,deepening the understanding of the toxic mechanisms of MA and KET,and providing a preliminary basis for the study of therapeutic regiments for acute toxicity of drug abuse.