Accelerated telomere shortening in peripheral blood lymphocytes after occupational polychlorinated biphenyls exposure

Polychlorinated biphenyls (PCBs) are organochlorine pollutants with a worldwide dissemination. We examined telomere length (TL) in peripheral blood cells of 207 individuals with a high body burden of PCBs due to occupational exposure in a transformer recycling company. Whereas TL in granulocytes was not affected, the age-adjusted TL in lymphocytes (∆TLLymph) of exposed individuals was significantly shorter than expected [−0.77 kb; 95 % confidence interval (CI) −0.9316; −0.6052; p = 0.0001]. PCB exposure did not affect lymphocyte numbers or T cell receptor excision circle (TREC) levels in T cells, suggesting that PCBs cause loss of telomeric DNA in T cells due to their metabolic activation and antigen-stimulated proliferation. In support of this hypothesis, blood plasma levels of PCB-exposed individuals inhibited expression of telomerase, the telomere elongating enzyme in vitro in antigen-specific T cell proliferation assays. 3-OH-CB28, a downstream metabolite of the lower chlorinated PCB-28 in PCB-exposed individuals (mean blood plasma concentration: 0.185 ± 0.68 ng/mL), inhibited telomerase gene expression within 48 h of incubation in lymphoproliferative assays starting at a concentration of 0.27–6.75 µg/mL and accelerated telomere shortening in long-term cell culture experiments. Accelerated telomere shortening due to PCB exposure may lead to limitations of cell renewal and clonal expansion of lymphocyte populations. As PCB-related immune dysfunctions have been linked to increased susceptibility to infectious diseases and increased risk of cancer, our data provide a possible explanation, for how PCBs could promote infections and cancer through limiting immune surveillance. Electronic supplementary material The online version of this article (doi:10.1007/s00204-016-1725-8) contains supplementary material, which is available to authorized users.


Telomerase expression and activity.
Proliferating T-cells were lysed in TRIzol reagent, and RNA was purified according to the manufacturer`s instructions (Invitrogen, Carlsbad, USA). All RNA samples were subjected to DNAse I treatment. cDNA was synthesized using random hexamers primer and Superscript III reverse transcriptase according to the manufacturer`s instructions (Invitrogen, Carlsbad, CA, USA). For quantitative analysis of htert mRNA expression, 60-90 ng cDNA was analyzed for each experiment using a sequence detector (7500 Fast Real-Time PCR System; Invitrogen) and TaqMan target mixes (Assay-on-Demand Gene expression reagents; Invitrogen). For the determination of telomerase activity the TRAPeze® RT Telomerase Detection Kit (Merck Millipore, Darmstadt, Germany) was used according to the manufacturer´s instructions. Cells were lysed by repeating freeze and thaw cycels in CHAPs buffer followed by 30 minutes of incubation on ice. After centrifugation at 16 000 g for 20 min at 4°C, aliquots of the supernatant were stored at -80°C. Protein concentration of extracts was determined with the DC Protein Assay (Bio-Rad). Using the ABI Prism 7500 Fast real time cycler (Invitrogen), samples were incubated for 30 min at 30°C and amplified in 45 PCR cycles for 15 s at 94° C, 1 min at 59°C and 10 s at 45°C. The threshold cycle values (C t ) were determined from semi-log amplification plots (log increase in fluorescence versus cycle number) and compared with standard curves generated from standard telomeric repeats provided with the kit. For the determination of telomerase activity in ddGTP or 3-OH-CB28 incubated whole cell lysates, preincubation time was extended to 120 min at 30°C. In ddGTP assays, standard curves from telomeric repeats generated in the presence of ddGTP were used.

MMqPCR
The average telomere length of Jurkat T-cells after different days of culture was estimated by monochrome multiplex Q-PCR as described. In brief, telomeres (T) and the beta-globin gene (S; single copy) were amplified on a MyIQ2 Two-color Realtime PCR Detection-System (Biorad).
Analysis was performed in duplicates and serial dilutions of standard DNAs were used to calculate the T/S ratio. Amplification efficiency for the telomere and the beta-globin was between 90% and 110% in each run. Specificity of the amplification was confirmed by melt-curve analysis.

Confocal Q-FISH
Telomere Q-FISH staining was performed as described previously. Cultured cells were frozen using standard procedures and all samples were processed in parallel. After thawing, cells were spun on microscope slides and fixed with formaldehyde (Sigma, US). After three additional washing steps, slides were dehydrated with ethanol and telomeres were stained with Cy3-(C3TA2) PNA (Panagene, South Corea) followed by six further washing steps. DNA counter staining was carried out using DAPI solution (Sigma, US) and TL analysis was performed within 48 h using a high-resolution LSM710 (Zeiss, Jena, Germany) confocal microscope. Images were captured using 63x optical magnification with additional 2x zoom and multi-tracking mode on 0.5 µm steps was used to acquire images of DAPI and Cy3 staining. Maximum projection of 5 single consecutive steps was carried out and acquired images were used for further digital image analysis. TL detection was carried out using Definiens software (Definiens, Germany). Nuclei and telomeres were detected based on the respective DAPI and Cy3 intensity. Mean nuclear background of the Cy3 staining nuclei was calculated and subtracted of each detected telomere within the respective nucleus. Median value of the telomere length per single detected nucleus was used for analysis. TL quantification is given in arbitrary units of fluorescence (a.u.).

Supplemental Figures:
A) Telomerase activity and expression of primary unstimulated and stimulated lymphocytes. PBMCs from healthy donors were stimulated with tetanus toxoid for 5 days and telomerase activity and expression were assessed as described.   A: Distribution of non-dioxin like (ndl) PCB congeners, with Sum of lower chlorinated (lc) PCB28 + PCB52 + PCB101 and the sum of all non-dioxin like PCB