| Objective:To investigate the body burdens of polycyclic aromatic hydrocarbons (PAHs) of pregnant women and fetus and the contribution of each polycyclic aromatic hydrocarbon to the total carcinogenesis of 7 PAHs, to research the genome-wide expression change of fetus caused by PAHs exposure during gestation period, analyze the correlation between PAHs levels and cancer-related gene expression, and evaluate the contribution of each kind of PAHs to gene expression, so that provide evidence for exploring the carcinogenesis mechanism of PAHs and provide reference for selection of environmental priority control pollutants and for maternal gestation healthcare and diseases prevention for the next generation.Methods:1. Questionnaire investigation on randomly selected and voluntarily participating pregnant women was conducted by face to face and maternal venous blood and cord blood were collected.2. Total 7 kinds of PAHs, which were Pyr, BaA, BbF, BkF, BaP, DBahA and BghiP, were detected by high-performance liquid chromatography (HPLC) after maternal blood and cord blood were collected from March to July in 2010. The cord serum PAHs levels were compared with the maternal serum levels and the contribution rate of each PAH to the total carcinogenesis of 7 PAHs in both maternal and cord blood was analyzed.3. The whole genome mRNA expression of different PAHs exposure groups were tested through both Affymetrix and Illumina genome-wide mRNA expression array and Real-time PCR validation was carried. 4. Simple correlation analysis was used to research the correlation of gene expression with levels of Pyr, BaA, BbF, BkF, BaP, DBahA, BghiP and some other factors such as newborn gender, gestation age, maternal passive smoking, foliate acid supplement, and multivariate linear stepwise regression was also conducted to select the key impact factors of each cancer-related gene expression change.5. Canonical correlation analysis was used to analyze not only the correlation of two groups of PAHs and six genes as a whole, also the relationship of each PAHs or gene expression with the whole group and the opposite group, so that the contribution of every PAHs to the whole six gene expression was estimated.Results:â… Body burden of PAHs and carcinogenesis contribution of each PAH of pregnant women and fetus1. The detection rate of Pyr was the highest which was 97.62% and 96.77% in maternal and cord blood, respectively. The detection rates of the other 6 PAHs were higher than 70% in both maternal and cord serum. The concentrations of Pyr, BaA, BaP and DBahA in cord serum were statistically higher than those in maternal serum (p<0.05), while the concentration of BbF was statistically lower than that in maternal serum (p<0.05).2. The contribution of BaP, BbF and DBahA were dominant in the total carcinogenesis of 7 PAHs, which were 36.6% and 38.6%,28.0% and 19.0%, 21.1% and 25.9% in maternal and cord serum, respectively.â…¡The whole genome mRNA expression change of fetus induced by gestation PAHs exposure1. According to the results of Illumina array, there're 660 gene probes had different expression between two groups with high and low cord sreum PAHs levels, and 342 probes showed upregulated expression and 318 probes showed downregulated expression.2. According to the results of Affymetrix array, there're 1847 gene probes had different expression among five groups with different cord sreum PAHs levels, and 998 probes showed upregulated expression and 849 probes showed downregulated expression.3. The main GOs with statistic significance that different expression genes attend included DNA damage repair, cell proliferation and differentiation, apoptosis regulation, regulation of signaling transduction, embryonic developmental growth, embryonic placenta development, regulation of neurogenesis, eye and embryonic cranial skeleton morphogenesis, regulation of T cell receptor signaling pathway, response to organic cyclic substance, and so on.4. The main pathways with statistic significance that different expression genes attend included olfactory transduction, taste transduction, MAPK signaling pathway, metabolic pathways, melanoma, thyroid cancer, pathways in cancer, cardiac muscle contraction, Insulin signaling pathway, neuroactive ligand-receptor interaction, cell adhesion molecules (CAMs), folate biosynthesis, biosynthesis of unsaturated fatty acids, and so on. It indicated that the effects of gestation PAHs exposure on fetus may involved in cancer, diabetes, cardiovasculare diseases, neurogenic diseases and development of taste and olfaction, etc.5. In the whole pathway-net made according to the interaction of pathways noted by the KEGG, the pathway in cancer standed in the main upstream position with 6 downstream pathways, and MAPK signaling pathway were the key final-effects pathway.â…¢Relationship between gestation PAHs exposure and mRNA expression of key genes1. According to the results of simple correlation analysis between gene expression and infants gender, maternal passive smoking, fried food taken and foliate acid supplement during pregnant period, maternal education, mother age and gestation age, no significant correlation was found.2. The simple correlation analysis between six genes expression and cord serum concentrations of Pyr, BaA, BbF, BkF, BaP, DBahA, BghiP showed that the mRNA expression of SNRPN increased with the increasing of cord serum concentrations of Pyr, BaA, BbF, BkF, DBahA and BghiP, that meant it showed upregulated expression with the PAHs exposure increasing; the mRNA expression of RPL27A was positively correlated with the concentrations of Pyr, BaA, BbF, BkF, BaP and BghiP; the mRNA expression of BMP1 was positively correlated with the concentration of BbF; the mRNA expression of NUDT2 was positively correlated with the concentration of BaA, BbF, BaP and DBahA; the mRNA expression of RAD54B was positively correlated with the concentration of BaP and BghiP; while the mRNA expression of E2F1 was positively correlated with the concentration of BbF, BaP, DBahA and BghiP, that was meant it showed down-regulated expression with the increasing of gestation PAHs exposure.3. The result of multivariate linear stepwise regression showed that cord serum BbF was the key factor that affected the mRNA expression of SNRPN and it significantly positively affected the mRNA expression of SNRPN (p=0.033); the mRNA expression of RPL27A was mainly positively affected by the level of cord serum BkF (p=0.001) and negatively affected by maternal education level (p=0.014), the effect of BkF was stronger than that of maternal education because of the absolute value of standard regression coefficient from BkF (b'=0.444) was bigger than that from maternal education (b'=-0.314); the mRNA expression of BMP1 was mainly positively affected by the level of cord serum Pyr (p=0.043); the mRNA expression of NUDT2 was mainly positively affected by the level of cord serum BaP (p=0.035); the mRNA expression of E2F1 was mainly negtively affected by the level of cord serum BaP (p=0.039); the mRNA expression of RAD54B was mainly positively affected by the level of cord serum BaP (p=0.004) and negtively affected by the level of cord serum BkF (p=0.029), the absolute value of regression coefficient from BaP (0.428) was bigger than that from BkF (-0.316), which meant that the effects of BaP on RAD54B mRNA expression was likely stronger than that of BkF.IV Comparison of the impacts on mRNA expression of every kinds of PAHs1 The correlation coefficient of the Canonical correlation variant W1 (which reflected the status of 6 genes expression) with BbF (0.5352) was the biggest one and the second and third was BkF (0.4456) and BaP (0.3867), respectively, which meant that the impact of BbF on the mRNA expression of six cancer-related genes ranked the first, then was that of BkF and BaP.2 The correlation coefficient of the Canonical correlation variant V1 (which reflected the level of 7 kinds of PAHs) with the mRNA expression of RPL27A was the biggest one, and the second and third was SNRPN and NUDT2, which indicated that the impacts of BbF on the mRNA expression of six cancer-related genes ranked the first, then was that of SNRPN and NUDT2. Conclusions:1. Several kinds of carcinogenic PAHs can penetrate the placenta and get into the body of fetus. The contributions to carcinogenesis risk of BaP, BbF and DBahA are bigger than the other kinds of PAHs detected.2. Gestation PAHs exposure can be involved in the mRNA expression change of many cancer-related genes, and increase the risk of tumor formation.3. SNRPN, RPL27A, BMP1, NUDT2 and E2F1 can be regarded as the candidate genes of the PAHs'target gene.4. Different kind of PAHs has effects on mRNA expression of different genes, which means they may have different mechanisms of adverse effects.5. BbF showed the most contribution on the mRNA expression change of the six cancer-related genes, and then BkF and BaP.6. It's feasible that discriminating the strengthen and contribution of carcinogenesis of every PAHs by the change of gene expression caused by each kinds of PAHs.
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