Long-term Persistent Organic Pollutants Exposure Induced Telomere Dysfunction And Lead To Senescence-associated Phenotype

Objective:Persistent organic pollutants?POPs?are the general term for refractory organic compounds that show long-range atmospheric transport,environmental persistence,and bioaccumulation.It has been reported that the accumulation of POPs could lead to many human diseases,including reproductive disorders,immune disorders,nervous system toxicity,which have attracted the worldwide attention.In the previous study,people lived POPs exposure region showed severe cell apoptosis,degenerative changes.In this study,we will investigate the effects of POPs exposure on chromosome stability,gemome modification,telomere dysfunction and inflammatory response systematically,and clarify the potential mechanism and signaling pathways of aging induced by POPs,providing basis and guidance for the intervention program of POPs induced aging diseases.Methods:?1?The sample collection areas were divided into the exposed area and the control area.The exposed area was the village near the e-waste area in Jinghai County,Tianjin and the unexposed area?control area?was the place that is 40 km away from e-waste area where people have similar overall environmental condition and personal life style,but have not participated in any e-waste disposing.Informed consent was obtained from all participants before taking any specimen.12 mL of peripheral blood?heparin anticoagulant,EDTA anticoagulant,procoagulant whole blood,4 mL respectively?was collected after routine physical examination from population of different regions.At the same time,questionnaires that consist of age,history of illness and childbearing history,etc.were collected,we will investigate the effects of POPs exposure on aging diseases such as hypertension,diabetes,autoimmune and reproductive diseases through statistical analysis.?2?Immunofluorescence?IF?technique and cytokinesis-block micronucleus?CBMN?assay were used to analyze the degree of DNA damage and genomic toxicity in peripheral blood.?3?Fluorescence in situ hybridization?FISH?and IF-FISH were used to analyze the telomere heterogeneity and telomere DNA damage induced by POPs exposure;to evaluate the effects of POPs exposure on telomere length and senescence,the telomere length of peripheral blood leukocytes was measured by telomere quantitative FISH?Q-FISH?,telomere DNA real-time PCR and telomeric DNA Southern Blot;to clarify the POPs components that were association with the human aging and degenerative diseases through the correlation analysis between telomere length and POPs components.?4?To clarify the effect of POPs exposure on gene epigenetic changes in the genome,especially in the subtelomere,DNA methylation level of peripheral blood genomic DNA and subtelomere DNA were examined by high resolution melting curve and reduced representation bisulfite sequencing?RRBS?,the expression of telomere RNA?TERRA?was detected by quantitative PCR and whether the telomere elongation replacement?ALT?was activated was defined by C-circle technique.?5?To define effect of POPs on human aging,ELISA and Real-time PCR were used to detect the expression of proteins and genes in p16^INK4a-Rb and p14^ARF-p53pathway.The levels of inflammation related cytokines including interleukin?IL?-6,IL-10,IL-2,tumor necrosis factor-??TNF-??,and nuclear factor-?B?NF-?B?in plasma were determined by enzyme-linked immunosorbent assays?ELISA?.Results:?1?Statistical results showed that percentage of individual with hypertension,diabetes and autoimmune diseases was significantly increased in POPs exposure population,so was the spontaneous abortion,indicating that long-term POPs exposure can induce the development of aging-related diseases.?2?The IF and micronucleus data showed observable increased 53BP1 foci??5dots?and micronucleus rate in the exposed individuals compared to control cells,indicating that long-term POPs exposure causes severe DNA damage and chromosomal instability.?3?FISH and IF-FISH data showed that POPs exposure could lead to significant telomere DNA damage,telomere loss and multi-telomere signal.FISH,quantitative PCR and Southern blot showed that telomere length of peripheral blood DNA in exposed individuals was significantly shorter than that in control individuals.Meanwhile,PBDE184,a POPs component,showed a significant negative correlation with telomere length.These results indicate that long-term exposure to POPs can result in telomere length shortening and telomere dysfunction.?4?HRM and RRBS assay showed that the methylation level of LINE-1 DNA and some subtelomeric DNA in population lived in exposure region were significantly decreased.The TERRA transcription level was notably increased in the individuals with the POPs exposure,especially on 20q and Xp/Yp chromosome.C-circle assay showed that POPs exposure may activate the alternative lengthening of telomeres?ALT?mechanism.These results suggested that long-term exposure to POPs can down-regulate DNA methylation at the genome-wide and in the subtelomeric regions while increase TERRA transcription and activates the ALT mechanism,which is consistent with our previously observation of increased telomeres heterogeneity and DNA homologous recombination in exposed individuals.?5?ELISA and quantitative PCR showed that the level of P53,Rb,P16^INK4aNK4a and P14^ARF in the plasma and the relative RNA level of p53 and Rb were dramatically increased in the exposed individuals suggesting that long-term POPs exposure activates senescence-related pathways.ELISA showed that the production of IL-6,IL-10,TNF-?and NF-?B in the plasma of POPs exposure population increased and the IL-2 decreased compared with the control population,indicating that the long-term exposure of POPs could cause the chronic inflammation.Conclusion:Long-term POPs exposure can lead to increased risk of the currence of aging and degeneration related diseases including hypertension,diabetes,autoimmune diseases,and affect the fertility of women.Long-term POPs exposure can lead to increased genomic instability,induce telomere length shortening and telomere dysfunction,resulting in the occurrence of individual senescence phenotypes.In addition,long-term POPs exposure can cause the change of DNA methylation levels,promoting the expression of TERRA,futher affecting the structure and function of telomere and inducing senescence,alterations in chromatin status could activate ALT and increase the risk of aging.At the same time,long-term exposure of POPs causes SASP and immune disorders with activating the NF-?B pathway,inducing chronic inflammatory state,which accelerate aging.The activation of p16^INK4a-Rb and p14^ARF-p53 pathways by long-term exposure of POPs confirms the occurrence of aging.