Effects And Related Mechanisms Of Polycyclic Aromatic Hydrocarbons And Phthalate Esters Co-exposure On Aging Signs

Polycyclic aromatic hydrocarbons（PAHs）and phthalate esters（PAEs）as persistent organic pollutants are widely co-existed in the environments.These two major kinds of parent compounds and their metabolites have been detected in various biological specimens,including blood,urine,human milk and seminal fluid.The levels of monohydroxylated PAHs（OH-PAHs）or PAEs metabolites in human body are now being considered as indicators of internal dose to reflect recent PAHs or PAEs exposure.Numerous studies showed the toxicities of individual PAHs or individual PAEs in the body,including lung function decline,inflammatory injure and reproductive toxicity.Owing to persistent co-exposure to multiple environmental pollutants,adverse health effects of co-exposure to environmental pollutants are hard to ascribe to one pollutant alone.Therefore,it is necessary for us to investigate adverse health effects of co-exposure of PAHs and PAEs to provide scientific basis for preventing and controlling environment-related diseases.Previous studies suggested that aging is a major risk factor for cardiovascular diseases,diabetes,neurodegenerative diseases and tumors.Exposure to PAHs and PAEs affect aging,including inflammatory response,oxidative stress,dysfunctions of telomere and mitochondria as well as lung function decline.However,the effects and relevant molecular mechanisms of PAHs and PAEs co-exposure on aging need to be further investigated.Thus,in the present study we firstly collected data of the participants from a panel study that performed in Wuhan city during the period of the winter（from December 2014 to February 2015）and the summer（from May to July 2015）.Each individual completed the questionnaires,participated the physical examination items（including white blood cell count,neutrophils,lymphocytes,platelet-lymphocyte ratio（PLR）,neutrophil-to-lymphocyte ratio（NLR）and systemic immune-inflammation index（SII））.Additionally,we measured the following indicators using the blood samples and three-consecutive days of urine samples collected during the period of the winter and summer seasons,including levels of OH-PAHs and PAEs metabolites,plasma levels of inflammatory indicators（interleukin-6（IL-6）and IL-8）,oxidative stress indicators（plasma levels of malondialdehyde（MDA）,glutathione peroxidase（GSH-Px）activity,oxygen radical absorbance capacity（ORAC）,hydroxyl radical antioxidant capacity（HORAC）,urinary levels of 8-hydroxy-2’-deoxyguanosine（8-OHdG））,aging indicators（relative telomere length（RTL）and mitochondrial DNA copy number（mtDNA-CN））and lung function parameters（forced expiratory volume in one second（FEV1）,forced vital capacity（FVC））.Then,we estimated the associations between exposure to individual PAHs or individual PAEs or PAHs and PAEs co-exposure and the indicators for inflammatory response,oxidative stress and aging.Considering that BaP and DEHP are the typical representative of PAHs and PAEs,respectively,we estimated benzo[a]pyrene-toxicity equivalent（BaP-TEQ）and daily intake（DI）of bis（2-ethylhexyl）phthalate（DEHP）by using urinary levels of OH-PAHs and DEHP metabolites,and further assessed influences of exposure of PAHs alone or PAEs alone or the combined effects of PAHs and PAEs on the body.Finally,in vitro was conducted to investigate the combined toxicity of DEHP and BaP in HepG2 cells.Part One Associations of urinary OH-PAHs and urinary PAEs metabolites with aging signsObjectives:Adverse health effects of PAHs and PAEs co-exposure were evaluated.Methods:A total of 120 subjects from a panel study were included in this analysis,the participants were obtained based on the 1240 subjects who were recruited from the two communities in Wuhan city by using a stratified cluster random sampling method.Each individual completed the questionnaires,took part in the physical examination and provided their blood and urine samples during the period of the winter（from December 2014 to February2015）and the summer（from May to July 2015）.High-performance liquid chromatography（HPLC）with a fluorescence detector,HPLC-tandem mass spectrometry and HPLC coupled with electrochemical detection were respectively conducted to determine the urinary levels of eight OH-PAHs and ten PAEs metabolites in three-consecutive days as well as 8-OHdG on the third day during the period of individual particulates with an aerodynamic diameter≤2.5μm(PM_2.5)measure.Peripheral blood genomic DNA of mtDNA-CN and RTL were analyzed by using real-time quantitative polymerase chain reaction.Measures of plasma MDA levels,GSH-Px activities,ORAC and HORAC were conducted by using the corresponding commercial kits.Moreover,plasma IL-6 and IL-8 levels were detected by using cytometric bead array.Linear mixed effect model and Bayesian kernel regression model（BKMR）were respectively used to assess the associations of exposure of individual PAHs or individual PAEs or PAHs and PAEs co-exposure with indicators of oxidative stress（MDA,GSH-Px,HORAC,ORAC or 8-OHdG）,inflammatory response（white blood cell count,neutrophils,lymphocytes,PLR,NLR,SII,IL-6 or IL-8）,aging（mtDNA-CN or RTL）and lung function parameters（FEV1,FVC or the FEV1/FVC ratio）.Subsequently,we calculated the BaP-TEQ and DI of DEHP based on the urinary levels of OH-PAHs and PAEs metabolites,and then assessed adverse health effects of BaP-TEQ alone or DI of DEHP alone or both of them on inflammatory response indicators,oxidative stress indicators or aging signs.Results:Linear mixed effect models indicated that there were negative associations between urinary OH-PAHs and RTL as well as between urinary levels of high molecular weight PAEs metabolites and mtDNA-CN.A one-unit increase in ln-transformed 3-day moving average value of urinary 2+3-hydroxyphenanthrene was associated with a 7.0%（95%CI:10.7%,3.2%）or 3.5%（95%CI:6.4%,0.58%）decrease in FEV1 or FVC.A one-unit increase in In-transformed 3-day moving average value of urinary monoethyl phthalate or mono（2-ethyl-5-oxohexyl）phthalate metabolites was associated with a 5.0%（95%CI:1.4%,8.6%）in FEV1 or 4.5%（95%CI:0.6%,8.6%）increase in FEV1.Results from BKMR models showed that eight urinary OH-PAHs had positive associations with indicators for inflammatory response（neutrophil count,NLR and SII）and oxidative stress（MDA,GSH-Px,HORAC,ORAC,8-OHdG）,but had negative associations with indicators for inflammatory response（IL-6 or IL-8）,aging（RTL）and lung function（FEV1 or FVC）;nine urinary PAEs metabolites had positive associations with inflammatory response indicators（lymphocyte counts or IL-6）or FVC,but had negative associations with indicators of oxidative stress（GSH-Px and HORAC）.Urinary PAEs metabolites antagonized either the positive effects of urinary OH-PAHs on inflammatory response indicators（increases in neutrophil counts,NLR or SII）and oxidative stress indicators（increases in GSH-Px or HORAC）or the negative effects of urinary OH-PAHs on inflammatory response indicators（decreased in IL-6 or IL-8）,aging signs（shorter in RTL and decreased FEV1/FVC）).We found that certain concentration of urinary PAEs metabolites attenuated the positive effects of urinary OH-PAHs levels on indicators of oxidative stress（increases in MDA,8-OHdG or ORAC）.Moreover,the estimated DI of DEHP enhanced either the positive effect of the estimated BaP-TEQ on MDA,GSH-Px or 8-OHdG or its negative effect on IL-6,IL-8 or RTL.There were interaction between the estimated DI of DEHP or estimated BaP-TEQ and inflammatory indicators（SII or PLR）or antioxidant indicators（HORAC or ORAC）.Conclusions:Exposure to certain concentration of PAEs may counteract PAHs-induced adverse effects in the body,including inflammatory response（increased neutrophils,NLR and SII）,lipid peroxidation（increased MDA content）,oxidative DNA damage（enhanced levels of urinary 8-OHdG）,activation of antioxidant defense system（increased GSH-Px,HORAC and ORAC）or alternation of aging signs（shorter RTL,decreased FEV1 and FVC）.The estimated DIs of DEHP and BaP-TEQ showed a positive interaction on HORAC or ORAC,but did a negative interaction on SII or PLR.The estimated DI of DEHP may enhance the positive effect of the estimated BaP-TEQ on mtDNA-CN and the negative effect of the estimated BaP-TEQ on RTL.Part Two ERK signal pathway involved in the co-exposure of DEHP and BaP-induced telomere dysfunction.Objectives:In vitro was conducted to investigate the potential mechanisms underlying the telomere dysfunction induced by DEHP alone or BaP alone or both of them in human hepatocellular carcinoma（HepG2）cells.Methods:We treated HepG2 cells with dimethyl sulphoxide（solvent control,final concentration<0.1%）,DEHP（62.50μM,125.00μM,250.00μM,500.00μM or 1000.00μM）alone,50.00μM BaP alone or 50.00μM BaP plus DEHP at the indicated concentration for 24h and 48h.We measured oxidative stress indicators（such as intracellular and mitochondrial reactive oxygen free radical（ROS）,HORAC,ORAC,GSH-Px and MDA）,mitochondrial function indicators(such as mitochondrial membrane potential,mtDNA-CN,mitochondrial Ca⁺² and mitochondrial mass),inflammatory response indicators（such as expression of IL-6and IL-8 at mRNA and protein levels,expression of SIRT1,telomerase reverse transcriptase（TERT）,TIN2-interacting protein 1（TPP1）,p38,p-p38,ERK and p-ERK proteins）.Furthermore,expression of sirtuin 1（SIRT1）,TERT and TPP1 proteins were measured in HepG2 cells treated with 10.00μM U0126（a p-ERK inhibitor）alone,500.00μM DEHP alone,50.00μM BaP alone or all of them at 24h and 48h.Results:In vitro showed that in HepG2 cells with DEHP alone at the certain concentrations or 50.00μM BaP alone or both of them,there were abnormal variances in the secretions of IL-6 and IL-8,MDA contents,GSH-Px activities,intracellular and mitochondrial ROS generation,mitochondrial Ca²⁺levels,mitochondrial mass,mitochondrial membrane potential and ATP levels.Besides,we found the up-regulated expression IL-6 and IL-8 at mRNA levels,increase in RTL and decrease in mtDNA-CN and cell proliferation rates.U0126（10.00μM）inhibited the up-regulated expression of TPP1 protein and down-regulated expression of TERT and SIRT1 proteins in HepG2 cells treated with 500.00μM DEHP plus50.00μM BaP at 48h.Conclusions:The certain concentrations（≥500.00μM）of DEHP with 50.00μM BaP at24h up-regulated expression of p-ERK protein,down-regulated expression of TERT protein and increased RTL in HepG2 cells.Inhibition of the ERK signaling pathway attenuated the combined effect of 500.00μM DEHP plus 50.00μM BaP on the down-regulated expression of TERT protein in HepG2 cells at 48h.