A Study On Mutagenicity Of Carbamazepine As Metabolically Activated By Human CYP Enzymes

BackgroundEpilepsy is one of the most common neurological disorders in the world,with an estimated 50 million people suffering from epilepsy worldwide.Carbamazepine(CBZ)is a tricyclic compound used as a traditional antiepileptic drug for the treatment of epilepsy and other neurological and psychiatric disorders.CBZ can be metabolized by cytochromeP450(CYP)enzymes to hydroxylated products.However,itremains unclear whether CBZ can be activated by CYP enzymes for mutagenic activities.ObjectivesTo explore the mutagenicity(gene and chromosome changes)related to the metabolism of mammalian cells by CBZ,the involved CYP enzymes and the mode of chromosomal damage.MethodsThe binding energy and conformation(determining a substrate potential)of CBZ for the active sites of human CYP1A1,CYP1A2,CYP1B1,CYP2B6,CYP2E1 and CYP3A4 were analyzed by using simulated molecular docking techniques,based on which further cytogenetic toxicity experiments using recominant cells lines expressing various human CYP enzymes were designed.Chinese hamster lung fibroblast(V79)-derived cell lines genetically engineered for the expression of various human CYP enzymes(their parental V79-Mz cell line was deficient in the activities of any CYPs,sulfotransferases(SULTs)and UDP-glucoronysyl transferases(UGTs)),human hepatoma(HepG2)cell line(with relatively low but inducible levels of CYP enzymes),and its derivative line C3A,which endogenously expresses various CYPs at elevated levels.Cell viability was determined by CCK-8 assay,while chromosomal damage was measured by micronucleus test,based on which the centromere contained in the micronuclei formed was analyzed by centromere protein(CENP)B immunofluorescence visualization.The content of-H2AX in the tested cells was analyzed by Western blot assay(its increase reflecting double-strand DNA breaks).Induction of gene mutations was analyzed by Pig-A assay.Results(1)Molecular docking results showed that the binding energy of CBZ with the active site of each human CYP enzyme,i.e.,CYP1A1,1A2,1B1,2B6,2E1 or CYP3A4,was negative,but regarding the distance between CBZ and the Fe ion in the active site of each CYP,only that for CYP2B6 and CYP2E1 was less than 6.0 (?)(supposed to permit electron transfer),while that for the other CYPs ranged from 8.0 to 10.1 (?).Therefore,molecular docking results suggest that human CYP2B6 and 2E1 are more likely to catalyze biotransformation of CBZ than other CYP enzymes.(2)Micronucleus experiments were conducted on recombinant V79-derived cell lines expressing each CYP enzyme.CBZ(exposing for 24 h)induced micronucleus formation in human CYP2B6-expressing cells at 10 μM and higher concentrations.However,in V79-Mz and other V79-derived cell lines expressing CYP1A1,CYP1A2,CYP1B1,CYP2E1 and CYP3A4 CBZ had no,or only weak,effect.Based on these results,C3A cells which endogenously expressed human CYP enzymes at relatively abundant levels were then used in the micronucleus test.CBZ(exposing for 72 h)significantly induced micronucleus formation(threshold concentration being 10 μM),and this effect was blocked by 1-aminopentriazole(ABT,a broad-spectrum CYP inhibitor)and ticlopidine(a specific inhibitor of CYP2B6).In addition,in HepG2 cells pretreated with CICTO(10 μM,activator of CAR)for 18 h,CBZ(exposing for 48 h)also induced micronucleus formation(threshold concentration being 10 μM),while in HepG2 cells pretreated with RIF(10 μM,PXR activator),CBZ only induced micronucleus at 40 μM,the highest test concentration,and no effect was observed in HepG2 cells pretreated with PCB126(40 nM,activator of AhR).(3)CENP-B immunofluorescence assay of CBZ-micronuclei formed in C3A cells was performed,and the results indicated that CBZ selectively induced CENP-B negative micronucleus,with a threshold concentration of 10 μM,in a concentration-dependent manner.Western blot assay of γ-H2AX in C3A cells showed that CBZ increased the level of γ-H2AX,,while of its level in HepG2 cells was unchanged.The above results suggested that CBZ has a purely clastogenic effect.PIG-A mutagenicity test showed that CBZ potently induced PIG-A mutations in C3A cells(threshold concentration 5 μM),while in HepG2 cells no effect of CBZ was observed,suggesting that CBZ has metabolic activation-dependent activity to induce gene mutations.ConclusionsCBZ can be metabolically activated by CYP2B6 for genotoxic potentials,and it may induce clastogenicity and gene mutations in mammalian(including human)cells at concentrations within its internal human exposure concentrations.