Study On Urinary Naphthol And 3-Phenanthrol As Exposure Biomarker Of Polycyclic Aromatic Hydrocarbons

There has been sufficient epidemiological evidence suggesting an etiological link between carcinogenic polycyclic aromatic hydrocarbons (PAHs) exposure and lung cancer risk in exposed workers.Chemicals produced in the coal-coking process has been classified as class 1 carcinogens by International Agency for Research on Cancer (IARC). Exposure biomarkers play an important roles in risk assessment process, such as dose-responds assessment and exposure assessment. Therefore, study on new biomarker of PAHs exposure will be beneficial for understanding of PAH carcinogenesis and improvement of risk assessment.In this cross-sectional molecular epidemiologically designed study, We establish an HPLC-MS/MS analysis method for simultaneous determination of urinary monohydroxy metabolites of PAHs. Base on this method, urinary monohydroxy metabolites were quantified among 147 PAHs exposed workers and 58 unexposed controls. We investigated the relationship between naphthol and 3-phenanthrol with exposure level, correlation with 1-hydroxypyrene. The relationship between naphthol and 3-phenanthrol with biomarkers of effect (Olive tail moment and genomic instability parameter) and biomarkers of susceptibility (genetic polymorphisms of metabolic enzymes) were investigated. The main results are as follows:Part 1. Naphthol and 3-phenanthrol determination method and study of exposure biomarkerUse naphthol-d8 as the internal standard, establish an HPLC-MS/MS method for the simultaneous determination of urinary monohydroxy metabolites of PAHs, namely 1-naphthol,2-naphthol and 3-phenanthrol, the limits of quantification(LOQ) was 0.2μg/L for 1-naphthol,2-naphthol and 1-pyrenol, LOQ for 3-phenanthrol was O.1μg/L. Internal standard calibration help the method has good reproducibility and precision, recovery of target compounds range from 97.4%to 103.0%, intra-and inter-batch precision was 1.4%-3.9% and 1.8%-5.8% respectively.Urinary monohydroxy metabolites quantified among 147 PAHs-exposed workers and 58 unexposed controls was corrected by urinary creatinine.The geometric mean of urinary naphthol and 3-phenanthrol was significant higher in PAHs-exposed workers than in unexposed workers. It was found that Urinary monohydroxy metabolites were well correlated with extra PAHs exposure level. Naphthol (Pearson's r=0.593, P<0.01) and 3-phenanthrol (Pearson's r=0.772, P<0.01) have an significant correlation with 1-hydroxypyrene. Cigarettes smoking was not a confounding factor to affect the concentration of 3-phenanthrol in urine.The results showed that urinary naphthol and 3-phenanthrol were valid exposure biomarker of PAHs, can serve as an useful complement of the common exposure biomarker 1-hydroxypyrene.Part 2. Association between naphthol 3-phenanthrol and effective biomarkers of genetic damage induced by PAHsUrinary 3-phenanthrol were divided into four group according to quartile. Chromosomal damage or instability status (present as the total number of micronuleus, nucleoplasmic bridge and nuclear bud in one thousand binucleated cells) was significiantly increased with the increased concentration of 3-phenanthrol. Olive tail moment was also increased with the increased concentration of 3-phenanthrol but not significiant as instability status. Significiant correlation was found in total population between 3-phenanthrol with instability status(r=0.644, P<0.01) and in PAHs-exposed worker(r=0.295, P<0.01). Correlation between 3-phenanthrol and Olive tail moment was found in total studied population (r=0.295, P<0.01).Urinary naphthol were divided into four group according to quartile. Chromosomal damage or instability status was significiantly increased with the increased concentration of naphthol, while Olive tail moment was not found this trend. Significiant correlation was found in total population between naphthol with instability status(r=0.450, P<0.01), ther is a certain correlation between naphthol with Olive tail moment in total population (r=0.207, P=0.005).Part 3. Association between genetic polymorphisms of metabolic enzymes and levels of naphthol and 3-phenanthrolThe gene encoding metabolic enzymes are highly polymorphic and can affect the metabolism of PAHs.We analysis the association between naphthol and 3-phenanthrol with common genetic polymorphisms of metabolic enzymes phaseⅠandⅡincluding cytochrome P450 1A1, cytochrome P450 2E1, microsomal epoxide hydrolase, glutathione S-transferase and NADP quinone oxidoreductasel.The enzymes are involving in the metabolic process of nathlene and phenanthrene.Among PAHs-exposed workers, there was a significant association between Val462 allele of CYP1A1 gene and increased urinary 3-phenanthrol level (P=0.035). We have not found significant association between 3-phenanthrol with polymorphism of phaseⅡenzymes.Among PAHs-exposed workers, the significant association was found between His113 allele on exon 3 of mEH gene and the level of naphthol was higher which has His/His genetype than which has Tyr/His genetype (P=0.015). However, among non PAHs-exposed workers, there was an association between His113 allele on exon 3 of mEH gene (P=0.049) and the naphthol level was lower which has His/His genetype than which has Tyr/His genetype. The different effects of genetic polymorphisms on naphthol level between PAHs-exposed workers and non-PAHs-exposed workers suggested the existence of genetic polymorphisms and gene-environment interactions. We have not found significant association between level of naphthol with polymorphism of phaseⅡenzymes.In summary, the HPLC-MS/MS method for simultaneous detecting urinary was established, The new new biomarker was developed and validated.We investigated the relationship between naphthol and 3-phenanthrol with environmental exposure level, and explored the factors of affecting their concentration.The results indicated that naphthol and 3-phenanthrol can reflecte the environmental exposure of PAHs well, can be used as new exposure biomarker. The study also explored the relationship between new exposure biomarker with effect biomarkers and genetic polymorphisms of metabolic enzymes, which further validated the effectiveness of naphthol and 3-phenanthrol as a new exposure biomarker.