Micronuclei Formation By Promutagens In Metabolism-Incompetent V79 Cells Interacting With Activation-proficient Cells In Various Experimental Settings

BackgroundThe biomembrane permeability of reactive metabolites formed outside of test cells may be uncertain,which will influence the genotoxic effect of a test compound on the target cells.The negatively charged sulfo-conjugates formed extracellularly may not readily enter target cells,and some metabolites formed by cytochrome P450(CYP)enzymes may not endure a process of transportation.Up to now the introduced metabolic activity to in vitro genotoxicity assays is generally by the rat hepatic microsome S9 mix,where the reactive metabolites formed outside of test cells may have varying transportability against biomembranes and endurance for a distance to cover,and the varied bioavailability of different metabolites may be influential on their toxic effects.Reports on this point are still lacking.ObjectivesThis study was aimed to uncover the variations in the genotoxic effects of various promutagens,from which reactive metabolites is formed in a genetically engineered cell line expressing a specific activating enzyne(s),in test cells deficient in specific activating enzymes but communicable to the activating enzyme-expressing cells,particularly in comparison to the straight effect of a promutagen in the metabolizing cells.Based on this investigation,the variability of the efficacy of the in vitro metabolic activating system(S9 mix)in the detection of various promutagens through genotoxicity assays could be understood,and the unique usefulness of genetically engineered mammalian cell lines expressing biotransformation enzymes in genotoxicity assays can be evidenced.MethodsSeveral common promutagens,including 1-methylpyrene[1-MP,activated sequentially by CYPs and sulfotransferases(SULTs)(twosteps)],1-hydroxymethylpyrene(1-HMP,the intermediate metabolite from 1-MP,a promutagen requiring further activation by SULTs),benzo[a]Pyrene(BaP,a promutagen activated CYP1A1 and 1B2),and aflatoxin B1(AFB1,a promutagen activated by CYP1A2 and 3A4),were used in V79-derived cell lines expressing biotransformation enzyme(s)specific for each promutagen and V79(control)cells communicable in different modes with the metabolizing cells,to determine the induction of micronuclei.The modes of communication between different cell populations included:control cells and metabolizing cells mixed at the 1:1 ratio,subatance communication between control cells and metabolizing cells permitable through 0.4?m pores and a 1 mm distance(with a transwell culturing system),and exposure oftest cells to medium transferred from incubation of metabolizing cells with a test chemical.The exposure/recovery regime for the miceonucleus test was 3 h/21 h.Western blot assay was employed to analyze the relative levels of each biotrasfornnation enzyme in each cell line.Results1-MP significantly induced micronuclei in a V79-derived cell line expressing both human CYP1A2 and SULT1A1(V79-hCYPlA2-hSULTlAl),in the presence of concentration dependence.1-MP demonstrated similar effects in the following experimental models:(1)V79-drived cell line expressing human CYP1A2(V79-hCYPIA2)and that expressing human SULT1A1(V79-hSULT1A1)mixed at the 1:1 ratio;(2)use of the transwell system described above for the communication between V79-hSULT1A1 and V79-hCYP1A2;and(3)V79-hSULT1A1 as test cells received medium transferred from a 3-h incubation of V79-hCYP1 A2 cell with I-MP.1-HMP also induced micronuclei significantly in V79-hSULT1A1 cells;however,the effect of 1-HMP in the following models was far weaker:(1)V79-Mz as test cells being communicable with V79-hSULT1A1 cells in the transwell system,(2)exposure of V79-Mz cells to medium transferred from the incubation of V79-hSULT1 A1 with 1-HMP.Both BaP and AFB1 induced micronuclei in V79 cells in the presence of S9 mix,with concentration dependence;in comparison,BaP induced an effect of similar potency in V79-derived cell line expressing human CYP1A1(V79-hCYP1A1),differently,AFB1 induced micronuclei in V79-hCYP1A2 cells at a significantly increased potency.Furthermore,BaP induced micronuclei in V79-Mz cells communicable with V79-hCYP1A1 cells at potency similar to that of the effect of BaP straightly in V79-hCYP1 A1 cells;while AFB1 did not induce micronuclei at the test concentrations in V79-Mz cell communicable with V79-hCYP1 A2 cells.ConclusionThe present study indicates that the chance for the ultimate toxicant of 1-MP(1-sulfoxymethylpyrene)to diffuse into target cells and exert toxic effects is far lower than 1-HMP,which is not charged;moreover,the reactive metabolite transformed from AFB1 may have limited bioavailability to target cells(far lower than that from BaP),in particular it may not endure the transportation crossing a distance.The results of this study support the unique importance of genetically engineered mammalian cell lines expressing biotransformation enzymes in genotoxicity assays.