Metabolic Activatlion-based Induction Of Chromosome Damage By 1-Methylpyrene In Mammalian Cells

Background1-Methylpyrene(1-MP)is representative of alkylated polycyclic aromatic hydrocarbons and a hepatocarcinogen.Its bioreactivity depends on sequential reactions catalysed by CYPs and sulfotransferases(SULTs),which lead to the formation of its electrophilic ultimate carcinogen.We have observed gene mutations and micronuclei formation by 1-MP in mammalian cell lines expressing both human CYP1A2(or 2E1)and SULT1A1,and 1-hydroxymethylpyrene(1-HMP)as an intermediate metabolite of 1-MP could be activated to genotoxic metabolites.However,the mode of 1-MP-induced chromosome damage(clastogenic or aneugenic effect)following metabolic activation remains unclear.ObjectiveThis study was aimed on the exploration of the mode of chromosome damage,breakage or loss,as induced by 1-MP under the action of metabolically activating enzymes,and its effects on the mitosis and relevant microtubule apparatuses.MethodsThe micronucleus test was employed to determine the chromosome damaging effect of 1-MP and 1-HMP in a humanhepatoma(HepG2)cell line,and its derivative which was proficient in biotransformation enzyme.The exposure/recovery regimes were designed according to the cell doubling cycle,two cycles for each regime:either a short exposure(0.25 cycle)followed by a long recovery(1.75 cycles),or a long exposure(2 cycles)without recovery(favorable for the detection of clastogens and aeugens,respectively).Based on the above experimental regimes,immunofluorescent staining of centromere protein B(CENPB)was used to discriminate the formed micronuclei as with or without centromere;this technique was also used to analyze the micronuclei formed in genetically engineered V79-derived cell line expressing both human CYP1A2 and SULT1A1(V79-hCYP1A2-hSULT1A1).Furthermore,immunofluorescent visualization of β-tubulin and γ-tubulin in metaphases of the test cells exposed to 1-MP and 1-HMP were performed to explore their effects on the morphology of centrioles and spindle body.ResultsUnder a short exposure/long recover schedule,1-MP and 1-HMP started to induce micronuclei formation in C3A cells at the concentration of 20 μM and 1 μM,respectively,while they did not induce micronuclei in HepG2 cells.Furthermore,the effects of both compounds were blocked by 1-aminobenzotriazole(ABT,60 μM,a CYP inhibitor)or pentachlorophenol(PCP,10 μM,a SULT1 inhibitor).1-MP induced micronuclei in V79-hCYP1A2-hSULT1A1 cells starting at the concentration of 20 μM,with most micronuclei free of centromere;while it induced micronuclei in V79-Mz only at concentrations>80 μM,most micronuclei containing centromeres.1-MP induced micronuclei in C3A cells at concentrations≥ 20 μM,while in HepG2 cells a concentration of at least 120 μM was required for the induction of micronuclei;in both cell lines,the formed micronuclei were unexceptionally centromere-negativeImmediately after a short exposure(without recovery),the effects of 1-MP and 1-HMP on the microtubule apparatuses in metaphases of C3A and V79-hCYP1 A2-hSULT1A1 cells were observed by immunofluorescent staining;the results indicated that both compounds induced morphological abnormalities in the spindle body and centrioles in metaphases at concentrations apparently lower than the thresholds for micronuclei formation described above.Under a long exposure(without recovery)regime,1-MP and 1-HMP induced micronuclei at the low concentrations that induced spindle body and centriole damage,and at the lowest concentrations(1 to 8 μM)1-MP induced micronuclei unexceptionally centromere-negative,which suggested that with metabolic activation 1-MP was potent for aneugenic relative to clastogenic activity.ConclusionFollowing metabolic activation,1-MP may cause clastogenic or aneugenic effect in mammalian cells,in response to different treatment regimes/concentrations.Under continuous exposure conditions,aneugenicity of 1-MP particularly potent;.While the short exposure/long recovery regime may direct 1-MP and 1-HMP exposing C3A cells toward exclusive clastogenic response,at relatively high concentrations.In view of the actual low level/continuous exposure to 1-MP,our results suggest that subsequent to metabolic activation 1-MP may have potent mitosis-disturbing(through interfering microtubule)and aneugenic activity.