Effects of Short-Term Exposure to Inhalable Particulate Matter on Telomere Length, Telomerase Expression, and Telomerase Methylation in Steel Workers

Background Shortened leukocyte telomere length (LTL) is a marker of cardiovascular risk that has been recently associated with long-term exposure to ambient particulate matter (PM). However, LTL is increased during acute inflammation and allows for rapid proliferation of inflammatory cells. Whether short-term exposure to proinflammatory exposures such as PM increases LTL has never been evaluated. Objectives We investigated the effects of acute exposure to metal-rich PM on blood LTL, as well as molecular mechanisms contributing to LTL regulation in a group of steel workers with high PM exposure. Methods We measured LTL, as well as mRNA expression and promoter DNA methylation of the telomerase catalytic enzyme gene [human telomerase reverse transcriptase (hTERT)] in blood samples obtained from 63 steel workers on the first day of a workweek (baseline) and after 3 days of work (postexposure). Results LTL was significantly increased in postexposure (mean ± SD, 1.43 ± 0.51) compared with baseline samples (1.23 ± 0.28, p-value < 0.001). Postexposure LTL was positively associated with PM10 (β = 0.30, p-value = 0.002 for 90th vs. 10th percentile exposure) and PM1 (β = 0.29, p-value = 0.042) exposure levels in regression models adjusting for multiple covariates. hTERT expression was lower in postexposure samples (1.31 ± 0.75) than at baseline (1.68 ± 0.86, p-value < 0.001), but the decrease in hTERT expression did not show a dose–response relationship with PM. We found no exposure-related differences in the methylation of any of the CpG sites investigated in the hTERT promoter. Conclusions Short-term exposure to PM caused a rapid increase in blood LTL. The LTL increase did not appear to be mediated by PM-related changes in hTERT expression and methylation.


Research
Epidemiological studies have consistently linked both short-and long-term exposure to ambient particulate matter (PM) with increased morbidity and mortality from cardio vascular disease (Brook et al. 2004;Dockery et al. 1993;Hassing et al. 2009). Increased oxidative stress and inflammation are two major mechanisms involved in mediating PM effects (Chuang et al. 2007;Schwartz 2001;Seaton et al. 1995). Shortening of blood leukocyte telo mere length (LTL) has been proposed as a marker of cumulative burden of oxidative stress and inflammation (Bekaert et al. 2007;Fitzpatrick et al. 2007). Recent human studies have shown that longterm exposure to air particles from vehicular traffic or related pollutants is associated with reduced LTL (Hoxha et al. 2009;McCracken et al. 2010). In addition, Valdes et al. (2005) demon strated a dose-dependent inverse relationship between shortened LTLs and packyears of smoking. The dose-dependent effects of smoking were replicated by Morla et al. (2006) in a cohort of male smokers with and without chronic obstructive pulmonary disease (COPD). However, experi mental studies show that during acute inflammation, telo mere length in inflammatory cells undergoes a transient increase, which is believed to contribute to the proliferative capacity and clonal expansion necessary to build up an efficient inflammatory response (Weng et al. 1997). These findings are consistent with in vitro results showing that LTL can increase at varying rates during cell division as a result of compensatory mecha nisms (Hodes et al. 2002). Taken together, these observations suggest that shortterm PM exposures might produce an acute increase in LTL, which may participate in sustaining the inflammatory mechanisms associated with PM health effects. Nonetheless, whether short-term PM exposure affects LTL in exposed humans has yet to be evaluated.
Telo meres are composed of repetitive G-rich sequences (TTAGGG) and an abundance of associated proteins that together form a dynamic cap that protects chromosome ends (Blackburn 2001). The progressive telo mere shortening associated with cell division is conducive to cell senescence and reduced proliferative capacity (de Lange 1998). Telomerase is a ribo nucleo protein that can synthesize telo mere repeats and thus compensate or reverse telo mere loss associated with cell division (Greider 1996). The telomerase complex consists of two essential components: a catalytic unit, telomerase reverse transcriptase (hTERT), and the ubiquitously expressed RNA template, hTR. A positive correlation has been found between the amount of hTERT mRNA and telomerase activity (Cong and Bacchetti 2000;Li et al. 2003;Yi et al. 1999). The level of hTERT mRNA expression has been indicated as a primary mechanism of telomerase regulation (Liu et al. 2004). The hTERT 5´ flanking region is located in a CpG island (Guilleret et al. 2002), which is highly methylated in normal tissues (Vera et al. 2008), thus suggesting that hTERT mRNA expression might be regulated by DNA methylation.
Work conditions may cause exposure to indoor PM levels that are considerably higher than outdoor ambient concentrations. In industrial settings, workers are usually assigned to specific job tasks that tend to be repeated regularly over time. Differences in work routines may thus determine wide and stable gradients of individual exposure to PM, even among workers in the same work facility. However, the regular weekly schedule made of working days and days of rest determines a cycle of exposures and washouts that can be exploited to evaluate potential short-term effects (Tarantini et al. 2009). Steel workers are exposed to high levels of airborne PM and have been shown to be at higher risk for cardio vascular disease (Andjelkovich et al. 1990). In a group of steel workers exposed to high levels of airborne PM, we measured LTL, as well as mRNA expression and promoter DNA methylation of hTERT, to identify the effects of short-term exposures on blood LTL.

Materials and Methods
Subjects and study design. In a steel production plant in Brescia in northern Italy, we recruited 63 male workers free of cardiopulmonary disease or cancer. All participants had been working in the current job position for at least 1 year and had worked in the plant for 16 ± 10.1 years (mean ± SD; range, 3-35 years). All participants had a rotating weekly schedule based on a 6-day rotation made of 4 days of work followed by 2 days off.
To investigate short-term effects of PM, we obtained blood samples at two different times: The baseline sample was collected in the morning of the first day of a working week (after 2 days off work) before the beginning of any work activity, and the post exposure sample was collected at the same hour on the fourth day of work, after 3 consecutive days of work. A selfadministered questionnaire was used to collect detailed information on lifestyle, drug use, medical conditions, body mass index (BMI), education, and residential history. Records from the factory adminis trative files were used to extract information on occupational history. Individual written informed consent and approval from the local institutional review board were obtained before the study.
Exposure assessment. Measures of PM with aerodynamic diameters < 10 μm (PM 10 ) and < 1 μm (PM 1 ) obtained in each of the 11 work areas of the steel production plant were used to estimate individual exposures. PM 10 and PM 1 were measured during the 3 days between the baseline and postexposure blood drawing using a GRIMM 1100 light-scattering dust analyzer (Grimm Technologies, Inc., Douglasville, GA, USA). During the 3 working days between the baseline and the post exposure blood collection, each of the study subjects recorded in a personal log the time he spent in each of the work areas. Individual exposure was calculated as the average of area-specific PM levels weighted by the time spent in each area.
DNA extraction. We used EDTA tubes to collect 7 mL whole blood that was immediately centrifuged on site at 2,500 rpm for 15 min. The buffy coat (400 μL) was transferred in a cryovial and stored at -20°C until DNA extraction. DNA was extracted using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA) following the manufacturer's instructions. Purified DNA was resuspended in the kit hydration solution, quantified, and stored at -20°C until use.
LTL measure ment by quantitative poly merase chain reaction (qPCR). We measured LTL on buffy coat DNA using the realtime quantitative PCR method developed by Cawthon (2002) with minor adaptations (Hoxha et al. 2009). This method measures the relative LTL in genomic DNA by determining the ratio of telo mere repeat copy number to single copy gene copy number (T/S ratio) in experimental samples relative to a reference sample. The telo mere and human β-globin PCR mix and the thermal cycling profile for both amplicons were previously described (Hoxha et al. 2009). We used pooled control DNA samples from this same study to create a fresh standard curve, ranging from 8 ng/μL to 0.25 ng/μL, in every telomere and human β-globin PCR run. All samples contained Escherichia coli DNA heated at 96°C for 10 min and cooled at room temperature. DNA sample (15 ng) was added to each reaction (final volume, 20 μL). All PCRs were performed in triplicate on a DNA Engine Thermal Cycler Chromo4 (Bio-Rad, Hercules, CA, USA).
The intra assay coefficient of variation for the T/S ratio in the present study was 8.1%. The reverse-transcriptase PCR reaction's mean efficiency for telo mere and human β-globin reactions were 99.5% and 97.5%, respectively.
RNA extraction and hTERT mRNA expression. We used the PAXgene Blood RNA Kit to isolate total RNA from whole blood stabilized in PAXgene Blood RNA tubes (both from Qiagen-PreAnalytix, Hombrechtikon, Switzerland). cDNA was synthesized using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA). hTERT expression was analyzed by real-time PCR using the Hs 00972650_m1 TaqMan gene expression assay (Applied Biosystems). All measurements were normalized to the expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase gene), which was measured using the Hs 99999905_m1 TaqMan gene expression assay (Applied Biosystems). All PCR runs were performed in triplicate on a 7900HT Fast Real-Time PCR System (Applied Biosystems). We used the thermal cycling profile and the PCR reaction mix components for both cDNA recom mended by the manufacturer (Applied Biosystems). Relative gene expression levels were determined on the basis of the ΔΔCT method according to Livak and Schmittgen (2001). Cycle threshold (CT) values obtained for hTERT were first normalized for each sample respective to the endogenous house keeping gene (CT hTERT − CT GAPDH = ΔCT sample ). Subsequently, the ΔCT value obtained was normalized to an external calibrator (ΔCT sample − ΔCT calibrator = ΔΔCT). A pooled study sample was used as a calibrator. Relative expression levels were calculated as 2 −ΔΔCT (Livak and Schmittgen 2001).
hTERT promoter methylation. We performed DNA methylation analyses on bisulfite-treated DNA using a highly quantitative analysis based on PCR pyro sequencing (Bollati et al. 2007;Pavanello et al. 2009). DNA (0.5 μg; concentration, 20 ng/μL) was treated using the EZ DNA Methylation-Gold kit (Zymo Research, Orange, CA, USA) according to the manufacturer's directions. Final elution was performed with 30 μL M-elution buffer. We developed the assay for telomerase methyla tion by locating the hTERT promoter using Genomatix software (Genomatix Software Inc., Ann Arbor, MI, USA) on chromosome 5 (start = 1346370; end = 1346970; length = 601 bases), and amplified the sequence between positions 1346370 and 1346678. In this assay, we measured percent 5-methylcytosine (5mC) at each of three individual CpG dinucleotide positions (position 1, 1346540; position 2, 1346542; position 3, 1346555) within a CpG island located in the hTERT promoter. A 50-μL PCR was carried out in 25 μL GoTaq Green Master mix (Promega), 10 pmol forward primer, 10 pmol reverse primer, 50 ng bisulfite-treated genomic DNA, and water. PCR cycling conditions were 95°C for 60 sec, 60°C for 60 sec, and 72°C for 60 sec for 50 cycles. PCR products were purified and sequenced by pyro sequencing as previously described (Bollati et al. 2007) using 0.3 μm sequencing primer. Primers for the assay were forward AGGTTTTGGATGTTAGGGATTTT, reverse-biotinylated CCACAAAACC CT AA AACTTCTCC, and sequencing GGAG TT GTTTGGGAAT.
Statistical analysis. Student's paired t-test was used to assess differences between baseline and post exposure measurements. We evaluated the association of the levels of PM (PM 10 and PM 1 ) with LTL, hTERT mRNA expression, or hTERT promoter methylation (separately at each of the three CpG positions) using simple linear regression models, as well as multivariable models adjusting for age, pack-years of smoking, and percent lympho cytes in the differential blood count. We fitted different sets of models using baseline measures, postexposure measures, or the difference between post exposure and baseline measures of the dependent variables (i.e., LTL, hTERT mRNA expression, or hTERT promoter methyl ation). In all models, regression coefficients were computed with ordinary least squares estimators. To compare the magnitude of the associations, volume 119 | number 5 | May 2011 • Environmental Health Perspectives we report regression coefficients expressing the increase in LTL corresponding to an increase in PM exposure equal to the difference between the 90th and 10th percentiles of exposure. Outliers were excluded from regression analysis by dropping observations with studentized residuals that were > 3 or < -3. We checked regression assumptions by performing diagnostic tests for each model, which included the Shapiro-Wilk test to verify normality of residuals and the White test to verify the homo geneity of variance of the residuals. A two-sided p-value < 0.05 was considered statistically significant. All statistical analyses were performed in SAS (version 9.1.3; SAS Institute Inc., Cary, NC, USA).

Results
Study subject characteristics. The mean age of the study subjects was 44 years, with a range of 27-55 years. The 25 study subjects (40%) who were current smokers reported smoking 13.0 ± 7.2 (mean ± SD) cigarettes every day. The mean ± SD BMI of the study participants was 26.5 ± 2.7 kg/m 2 . The average levels of exposure estimated for each subject during the 3 days between the baseline and post exposure blood draws were 262 ± 272 μg/m 3 (range, 74-1,220) for PM 10 and 8.0 ± 7.7 μg/m 3 range, 1.7-30.5) for PM 1 . PM 10 and PM 1 exposure levels were highly correlated (r 2 = 0.82).
Blood leukocyte counts. Table 1 shows the mean values, SDs, and range of the variables measured on the baseline and post exposure samples. Differential blood leuko cyte counts showed a moderate, non statistically significant increase in the proportion of lymphocytes in the post exposure samples (n = 62; 32.0 ± 7.6%) compared with the baseline samples (n = 62; 30.7 ± 6.7%; p = 0.069). We found no differences in total white blood cell number, percent granulocytes, and percent monocytes (Table 1).
PM exposure and LTL. LTL showed a significant increase from baseline (n = 57; 1.23 ± 0.28 T/S relative units) to post exposure (n = 57; 1.43 ± 0.51; p < 0.001; Table 1). Figure 1 shows the changes of individuals' LTL between the first day of a workweek (baseline) and after 3 days of work (postexposure). LTL was significantly increased both in current smokers (n = 21; baseline: 1.26 ± 0.30 T/S relative units; post exposure: 1.5 ± 0.58; p < 0.0001) and in non smokers (n = 36; baseline: 1.21 ± 0.27; post exposure: 1.39 ± 0.52; p = 0.002). As a sensitivity analysis, we repeated the baseline versus postexposure comparison after excluding the three subjects with the highest post exposure LTL values and found that the increase in LTL was still highly significant (baseline: 1.22 ± 0.28; post exposure: 1.34 ± 0.35; p = 0.001).
The levels of individual exposure to PM during the 3 days between the baseline and post exposure blood draws were significantly associated with post exposure LTL in unadjusted regression analysis (for PM 10 , β = 0.26, p = 0.002; for PM 1 , β = 0.25, p = 0.037), whereas we found no significant associations with baseline LTL (for PM 10 , β = 0.06, p = 0.18; for PM 1 , β = 0.02, p = 0.71; Table 2). Consequently, levels of individual exposure to PM were associated with the difference in LTL between baseline and post exposure samples (for PM 10 , β = 0.20, p = 0.003; for PM 1 , β = 0.22, p = 0.016). The results obtained from unadjusted regression models were similar to the results from models adjusted for age, BMI, pack-years of smoking, and percent lymphocytes (Table 2).
PM exposure and hTERT mRNA expres sion. We measured blood mRNA expression of hTERT to gain information on whether the PM-related increase in LTL was produced by telomerase activation. All the blood samples with valid measures had detectable levels of hTERT mRNA. hTERT mRNA was significantly decreased in post exposure samples (n = 58; mean ± SD, 1.31 ± 0.75) compared with baseline samples (n = 58; 1.68 ± 0.86; p < 0.001; Table 2). However, the levels of individual exposure to PM during the 3 days between the baseline and post exposure blood draws showed no significant associations with hTERT mRNA expression in either the baseline or post exposure samples. Also, PM exposure was not associated with the difference in hTERT mRNA levels between the baseline and post exposure samples (Table 2).

Discussion
In the present study of workers in a steel plant with well-characterized measures of exposure to a wide range of PM levels, we found a significant, dose-related increase in LTL after short-term exposure to PM. However, the PM-related increase in LTL did not appear to be explained by modifications of hTERT mRNA expression and DNA methyla tion. The finding of increased LTL in the present study is in contrast with previous reports on longterm PM exposure and LTL. McCracken et al. (2010) reported an inverse association between 1-year exposure levels to black carbon, a tracer of PM from vehicular traffic, and blood LTL in a cohort of elderly individuals in eastern Massachusetts. Hoxha et al. (2009) showed that blood LTL was shorter in police officers with long-term exposure to traffic pollutants than in office workers. The authors speculated that long-term exposure to PM may cause shortened LTL through PM-induced oxidative stress, to which the G-rich structure of telo meres are more sensitive than is normal genomic DNA.
Several experimental in vitro models have shown that during acute inflammation, which is a central process in mediating health effects from short-term PM exposure (Brook et al. 2004;Seaton et al. 1995), telo mere length increases in inflammatory cells (Weng et al. 1997). These experimental studies have indicated telomerase activation as a potential mechanism determining telo mere elongation during acute inflammation (Weng et al. 1995). We therefore further examined hTERT mRNA expression and its promoter methylation levels. However, our results do not support a major role for hTERT expression in determining increased LTL in human subjects exposed to PM. In fact, we found lower hTERT mRNA levels in post exposure samples, which might reflect inactivation by negative feedback of telomerase in the presence of long LTL. However, because the hTERT protein is also post transcriptionally regulated (Liu et al. 2001), and we did not have a direct measure of telomerase activity in our study, we cannot exclude that telomerase activation had a role in determining increased LTL.
In addition to telomerase, the set of proteins that are associated with telo meres and can affect telo mere length has been shown to be extremely complex, and the precise molecular mechanisms that regulate telo mere length in lymphocytes or other cell types is not fully understood (Hodes et al. 2002). We did not find consistent associations of PM exposure with hTERT mRNA expression and methylation. It is worth noting that previous investigations correlating hTERT methylation with its mRNA expression have shown both positive and negative associations (Dessain et al. 2000;Guilleret et al. 2002;Lopatina et al. 2003;Shin et al. 2003). Liu et al. (2004) suggested that those inconsistent correlations may be due to the involvement of a large variety of transcription factors interacting with the hTERT promoter. Future studies on PM effects should take into account the Table 2. Association of PM 10 and PM 1 exposure with LTL and hTERT mRNA expression measured in foundry workers at baseline and post exposure.

Unadjusted
Adjusted a Dependent variable Exposure  For inflammatory cells, the ability to under go extensive cell division and clonal expansion is crucial for effectively generating an inflammatory response (Hodes et al. 2002). Cells with shortened telo meres lose their ability to divide and become senescent or undergo apoptosis (Blackburn 2001). In contrast, longer telo meres guarantee the maintenance of cell capacity for rapid proliferation (Hodes et al. 2002). These mechanisms have been shown extensively in lympho cytic subpopulations involved in acute inflammation, including those in peripheral blood (Norrback et al. 1996;Weng et al. 1998). Clonal expansion of circulating leuko cytes, which occurs rapidly in subpopulations with longer telomeres, is expected to lead these subpopulations to be more represented in peripheral blood and thus result in an increase in average LTL (Hodes et al. 2002). In our data, we found a moderate, non significant increase in the proportion of circulating blood lymphocytes between baseline and post exposure samples. This increase was not as sharp as that observed for LTL, as expected if only specific lymphocytic subpopulations, such as subpopulations of T or B cells, were to proliferate (Hodes et al. 2002;Weng et al. 1998). Alternatively, migration of less mature leukocytes-which have under gone a lower number of cell divisions and are expected to have longer telo meresfrom the bone marrow into the bloodstream might have contributed to increased LTL in post exposure samples. In fact, previous studies have shown that systemic inflammatory responses to PM exposure are associated with the presence of less mature leukocytes in peripheral blood (Suwa et al. 2002;Tan et al. 2000;Terashima et al. 1997). Future studies in PM-exposed subjects should measure LTL after separation of specific leukocyte subpopulations based on their functions and maturity.
Our study design included measurements of LTL, hTERT mRNA expression, and hTERT methylation in blood leuko cyte samples taken on the first and fourth day of a workweek. The two samples were collected to identify short-term fluctuations associated with the weekly cycle of exposure due to working in the plant during the week, followed by exposure cessation during the 2 days off between consecutive weeks. We investigated a population with well-characterized exposure that also allowed for contrasting subjects over a wide range of different exposure levels. Our study was based on subjects working in several work areas of the same factory and did not include a different population of subjects without a specific condition of exposure to inhaled pollutants. However, our primary evaluation of the short-term effects of PM exposure was based on a comparison of paired baseline and post exposure samples from the same subjects, in which each subject served as his own control. Moreover, in the regression models evaluating the dose-response relation ships of PM exposure levels with baseline or post exposure measures of LTL, hTERT mRNA levels, and hTERT methylation, we controlled for potential confounders by fitting multi variable models that included as independent variables several individual characteristics.
Limiting our investigation to individuals working in the same facility avoided potential concerns that are related to the selection of external control subjects, who might have differed from the exposed population in terms of socio economic factors and other charac teristics determining hiring into the plant (Pearce et al. 2007). Nonetheless, the differences in the personal levels of exposure in our study group were large, providing sufficient contrast for identifying exposure-related associations. For example, the lowest level of PM 10 observed in our study population (74 μg/m 3 ) was only marginally higher than ambient PM 10 levels measured in the geographic area in which the plant is located [average annual ambient PM 10 levels between 41 and 57 μg/m 3 were recorded in the year of the study by different ambient monitoring stations in the Brescia area (Anselmi 2006)], whereas the highest level was 1,220 μg/m 3 . Workers in foundries may have additional exposures in addition to PM, including polycyclic aromatic hydrocarbons (Mirer 1998), carbon monoxide (Lewis et al. 1992), and non ionizing radiation (Gomes et al. 2002). Although the participants in our study were in a modern steel-production facility with state-of-the-art systems for chemical and physical exposure reduction, we cannot exclude that these exposures might have contributed to the observed associations.
We designed our study to take advantage of temporal short-term variations in the exposure due to the weekly cycle of days of work and days off. However, we acknowledge that factors other than the exposure to airborne PM that under go variations over a week might have influenced the results. For instance, smoking patterns might vary between working and leisure time. We showed that the post exposure increase in LTL was present both in current smokers and in non smokers, thus limiting the odds that our findings were driven by recent patterns of active smoking. However, we cannot exclude that other unmeasured risk factors, including recent exposure to passive smoking, influenced our results.

Conclusions
Our study showed an increase in blood LTL associated with short-term exposure to PM in a group of steel workers. We did not find evidence that the increase in LTL was mediated by changes in hTERT mRNA expression and hTERT methylation. Future studies are warranted to clarify the mechanisms under lying the increase in LTL induced by short-term PM exposure, as well as its potential roles in mediating PM effects.