Phenotypes Of Genomic Instability Induced By Polycyclic Aromatic Hydrocarbons And Related Genetic Polymorphisms

Chemicals produced in the coal-coking process has been classified as class I carcinogens by IARC.The product coke is formed by blending and heating suitable grades of coals to 1000-1400℃in the absence of oxygen.Coking workers are regularly exposed to coke oven emissions,which are mainly comprised of polycyclic aromatic hydrocarbons(PAHs).Epidemiological studies have shown that workers with a long-term exposure to PAHs had a significantly higher risk of lung cancer. Lung cancer of coke-oven workers has been classified as one of the eight prescribed occupational cancers in China,and its incidence rate was about 10 times of that of the general population.Therefore,lung cancer of PAHs workers is still a critical issue in the field of prevention and control of occupational cancers in our country.An early key event in carcinogenesis is the induction of genomic instability, which facilitates a proliferative cell to progress into a cancer cell.There is strong evidence that micronucleus(MN) frequency in peripheral blood lymphocytes is predictive marker of cancer risk,and increased levels of MNi formation are associated with early events in carcinogenesis.However,its complicated formation mechanisms, relatively high baseline make it too difficult to estimate the genomic damage induced by chemicals.Over the past decades,Cytokinesis-block micronucleus(CBMN) test has evolved into a comprehensive method for measuring DNA damage,cytostasis and cytotoxicity,which called Cytokinesis-block micronucleus cytome assays.This method is now also to score Nucleoplasmic bridge(NPB),a biomarker of dicentric chromosomes resulting from telomere end-fusions or DNA misrepair,and to score nuclear bud(NBUD),a biomarker of gene amplification.The "cytome" concept implies that every cell in the system studied is scored cytologically for its viability status(necrosis,apoptosis),its mitotic status(monoucleated,bionucleated, multinucleated) and its chromosomal damage or instability status(presence of MNi, NPBs,NBUDs and number of centromere probe signals among nuclei or MNi if such molecular tools are used in combination with the assay).Based on the new effect biomarkers of genomic instability induced by PAHs,it can help us to elucidate the carcinogensis of PAHs,improve the accuracy of risk assessment on PAHs.In this cross-sectional molecular epidemiologically designed study,NPBs and NBUDs were used as the effect biomarkers of the genomic instability in peripheral blood lymphocytes among 158 PAH-exposed workers and 69 unexposed controls by the CBMN test.For multivariate analysis,the NPBs and NBUDs data were sqrttransformed to normalize the variance.One-way ANOVA analysis was used to compare the NPBs or NBUDs' difference in different environmental exposure levels, different coking history levels and different age levels.The differences in sqrt-transformed NPBs or NBUDs data between each genotype of related genes were analyzed with multivariate analysis of covariance with adjustment for age,gender,and cigarettes per day in PAHs exposed workers and controls separately.The MTHFR haplotypes were estimated by Bayesian statistical method with the PHASE software. Indicators of genomic instability found in epidemiological study were validated in cell lines which benzo[a]pyrene can be metabolized in vitro.DNA double strand breaks (DSBs) were test byγ-H2AX foci formation.Part 1.Relationiship between biomarkers of genomic instability induced by PAHs and exposure levelsWe found that NPBs and NBUDs were significantly higher in PAH-exposed workers than in the controls(9.41±3.73,7.13±4.01 versus 1.88±1.49,2.20±1.73, respectively;P＜0.001 for both comparisons) in a dose-dependent manner.The dose-dependent increase was also observed by integrating variables of MNi,NPBs and NBUDs,although that for MNi not found in the same study.In all 227 subjects,a significantly positive correlation was found between urinary 1-OHP concentrations and the frequency of NPBs(Pearson's r = 0.741,P＜0.001) and NBUDs(Pearson's r = 0.64,P＜0.001).Combining with principal component analysis,it revealed that NPBs and NBUDs were more susceptible to PAHs exposure.Compared with male PAH-exposed workers,female workers had less NPBs or NBUDs.NBUDs were found higher in workers with longer coke-working history(P=0.01).No effects of age, smoking and alcohol using were found on frequencies of NPBs or NBUDs among PAH-exposed workers.Part 2.Association study between phenotypes of genomic instability and related genetic polymorphismMultivariate analysis of covariance revealed that PAHs exposed workers with the MTHFR 677 TT and CT-TT genotypes had significantly higher NPBs frequency(9.79±0.49,p=0.03 and 9.75±0.34,p=0.03) than those with CC genotype(7.97±0.56) with adjustment for covariates.Further haplotypes analysis of the MTHFR 677-1298, PAHs exposed workers carrying CC,TA,TC haplotype had significant higher NPBs frequency than those with CA(P = 0.03,P = 0.005 and P = 0.002 respectively).No significant association between C677T and A1298C polymorphism and NBUDs frequency was found in both groups.Based on haplotype analysis of MTHFR 677-1298,subjects carrying TA haplotype had significant higher NBUDs frequency than those with CA(P = 0.02) in PAHs exposed workers.No significant association between MTHFR gene haplotype and NPBs/NBUDs frequencies among controls was found.A DNA double-strand break(DSB) is a critical lesion that can promote genomic instability.Eukaryotic cells have developed two pathways to repair DNA DSBs, including the homologous recombination(HR) and the non-homologous end-joining (NHEJ) pathways.We performed genetic association studies in the PAH-exposed population study analyzing polymorphisms in genes involved in HR(NBS1,MRE11, RAD50,RAD52,RAD54L,RAD54B,XRCC2 and XRCC3) and NHEJ(XRCC4,LIG4 and XRCC5).In the single locus analysis,elevated NPB frequency statistically significantly associated with NBS1 rs1805794,rs1805800,MRE11 rs13447623, RAD52 rs1051672,RAD54B rs3019148(all P＜0.05),and elevated NBUD frequencies significantly associated with MRE11 rs 1014666,rs 11020799,rs 13447623, rs622961 and RAD54L rs10789488(all P＜0.05) in the HR pathway.In the NHEJ pathway,XRCC4 rs1382373,rs1478485,rs4703951 and XRCC5 rs10182201, rs3821107,rs828699(all P＜0.05)associated with NPB frequencies;LIG4 rs1805388 and XRCC4 rs4703951(all P＜0.05)associated with NBUD frequencies.Part 3.Genomic instability induced by benzo[a]pyreneBenzo[a]pyrene(B[a]P) can be activated in 16HBE-CYP1A1 cells which are human bronchial epithelial cell with CYP1A1 transformed.The vector control cell is 16HBEV in the study.Genomic instability induced by benzo[a]pyrene was tested by cytokinesis-block micronucleus cytome assays.DSBs were detected byγ-H2AX foci formation.After B[a]P treatment for 24h,higher concentration induced moreγ-H2AX foci in 16HBE-CYP1A1 and 16HBEV cells,but theγ-H2AX loci were less in 16HBEV cells than 16HBE-CYP1A1 cells at the same B[a]P treatment. Cytotoxicity of B[a]P tested by CBMN cytome assays,the values of binucleated cells (BNC) ratio and nuclear division index(NDI) decreased and those of necrosis increased in a dose-dependent manner in 16HBE-CYP1A1 cells.Apoptotic cells were increased at lower dose of B[a]P and rarely observed at the highest dose of B[a]P in 16HBE-CYP1A1 cells.In 16HBEV cells,no significant BNC ratio changed in lower than 10 taM B[a]P treatment;it was decreased 40%at 20μM group.NDI was decreased at higher than 10μM B[a]P treated 16HBEV cells.Necrosis and apoptosis increased in a dose-dependent manner lower than 10μM B[a]P treated 16HBEV cells; apoptotic cells were decreased to normal level at 20μM B[a]P treatment.The genomic instability was assessed by the frequencies of MNi,NPBs and NBUDs.NPBs and NBUDs increased in a dose-dependent manner,however,MNi dose-relationship only last to 10μM B[a]P treated group in 16HBE-CYP 1A1 cells.In 16HBEV cells,frequencies of MNi,NPBs and NBUDs were increased,but no significant difference of them was found between higher than 5 tam B[a]P treated groups.Those indicators of genomic instability were increased may be induced by DSBs and inhibition of apoptosis pathway. In summary,genomic instability induced by PAHs is investigated logically in both of population-based and laboratory studies.In PAH-exposed population,NPBs and NBUDs can be taken as effect biomarkers for chromosomal instability, polymorphism of MTHFR and DSBs repair genes are associated to biomarkers of genomic instability among PAHs-exposed workers.Based on laboratory study, CYP1A1 is key metabolized enzyme of B[a]P;CBMN cytome assays can be used to analyze cytotoxicity and genotoxicity of B[a]P comprehensively,validate the dose-effect relationship between PAHs and NPBs/NBUDs were validated in vitro, which found in population study.The genomic instability formation may be associated to DNA double strand breaks and inhibition of apoptosis pathway.These findings suggest that genomic instability play an important role in PAHs carcinogenesis,and comprehensive biomarkers can be applied to biomonitor exposed population.