The enzyme telomerase reverse transcriptase (TERT) is a cellular ribonucleoprotein with reverse transcriptase activity responsible for the maintenance of telomere length (Aubert and Lansdorp 2008). The main function of TERT is the addition of the hexamaric guanine-rich repetitive sequence (5′-TTAGGG-3′) to the chromosomal ends to compensate for the progressive loss of telomeric sequence, thereby maintaining chromosomal integrity and genetic stability (Aubert and Lansdorp 2008). Germ cells, certain white blood cells (e.g., lymphocyte), and cancer cells have active telomerase complexes, which make them relatively long-lived compared to other cell types (Finkel et al. 2007). In contrast, all other somatic cell types have a more finite life span that is regulated at the cell level; telomere erosion leads to loss of approximately 50–200 base pairs (bps) of telomeric DNA at each cell division until replicative senescence, a state of irreversible cell growth arrest and apoptosis, is reached (Chiu et al., 1997). The process of telomere-shortening can be accelerated by increased oxidative stress (von Zglinicki 2002). Independent of aging, telomere shortening is also associated with several health outcomes such as cardiovascular diseases (Haycock et al. 2014), atherosclerosis (Samani et al. 2001), hypertension (Demissie et al. 2006), diabetes (Zee et al. 2010), and mortality (Weischer et al. 2012).

Antimony is a silvery white metal that is commonly found within the Earth’s crust; it exists in either a trivalent or pentavalent state (ATSDR 1992). Antimony is a persistent, bio-accumulative, and toxic chemical. Exposure to and toxicity from antimony may arise due to occupational exposure, domestic use, or when it is used as a medical therapy (Sundar and Chakravarty 2010). Antimony is used to treat parasitic diseases such as leishmaniasis and schistosomiasis. Antimony is also used as a fire-retardant for plastics, and in textiles, rubber, adhesives, pigments in paints, lead storage batteries, pipe metals, semiconductors, and pewter (ATSDR 1992). Antimony is released into air and water from coal-burning power plants, incinerators, mines, and industrial facilities. In urban areas, the primary sources of exposure are likely to be vehicle exhaust and industrial emissions. The general population is constantly exposed to low levels of antimony in the environment through food, air, and drinking water (ATSDR 1992). People may also be exposed by skin contact with soil, water and other substances that contain antimony (ATSDR 1992).

The absorption, distribution, and excretion of antimony can vary depending on its oxidation state, with urinary excretion appearing to be greater for pentavalent antimony compounds than for trivalent compounds (Elinder and Friberg 1986). An elimination half-life of approximately 95 hours has been estimated after occupational exposures (Kentner et al. 1995). Human health effects from antimony at low environmental doses are not well characterized, although respiratory and cardiovascular effects have been reported (ATSDR 1992; Sundar and Chkravarty 2010). Studies have shown that most of the antimony that enters the body concentrates in the liver, lungs, intestines, and spleen (ATSDR 1992). It also rapidly accumulates in blood cells following in vivo administration (Winship 1987). Several in vitro studies report that antimony generates reactive oxygen species (ROS) and activates caspase-3 leading to cell apoptosis in human leukocyte cell lines (Lecureur et al. 2000a, b; Losler et al. 2009; Wyllie and Fairlamb 2006).

In this study, we conducted a cross-sectional analysis to examine the association of urinary antimony concentrations with leukocyte telomere length (LTL) in a representative sample of US adults (20 years of age and older) who participated in the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2002.

The geometric mean (SE) LTL of the study population (n=2307) was 1.03 (0.01). The weighted distributions of study population characteristics are shown in Table 1. Women represented slightly over 51% of the sample; the geometric mean age of participants was approximately 43 years old. The geometric mean (SE) of urinary antimony was 0.12 ng/mL (0.0003 ng/mL).

Table 2 shows the results of the multivariable linear regression. Briefly, in all three models, urinary antimony was statistically significantly associated with shorter LTL. Individuals in the 3^rd and 4^th quartiles of urinary antimony had statistically significantly shorter LTL (−4.78%, 95% CI: −8.42, −0.90; and −6.11, 95% CI: −11.04, −1.00, respectively) compared to the lowest referent quartile with evidence of a dose-response (p trend = 0.03) (Table 2 model 3). Furthermore, analyses using restricted cubic spline confirmed the dose-relationship between urinary antimony and LTL (Supplemental Figure 1a). Sensitivity analyses using antimony standardized by creatinine (expressed as μg/g of creatinine) showed similar dose-response relationships (Supplemental Figure 2a).

Analyses stratified by age group indicated that the statistically significantly association of shorter LTL with antimony were driven by middle aged (40–59 years) and older (60–85 years) adult groups. Conversely, in young adults (20–39 years) there was a statistically significantly higher LTL in individuals in the 2^nd antimony quartile compared to the referent quartile (Table 2). Figure 1 summarize the LTL percent difference by antimony quartile for all participants and by age group. Sensitivity analyses including lymphocytes and granulocytes proportion in whole blood yielded results similar to those from the primary analyses (data not shown). Further analyses using restricted cubic spline confirmed the dose-response relationship between urinary antimony and LTL in the different age groups (Supplemental Figures 1b–d). Sensitivity analyses using antimony standardize by creatinine (expressed as μg/g of creatinine) showed similar dose-relationship in the different age groups (Supplemental Figures 2b–d).

To our knowledge, this is the first study reporting on urinary antimony and leukocyte telomere length. In this general population study, we found that environmental exposure to urinary antimony was associated with shorter LTL. Specifically, participants in the 3^rd and 4^th quartiles of urinary antimony had a decreased leukocyte telomere length of 4.78% and 6.11%, respectively, compared to those in the referent lowest quartile. Telomere erosion by age (as calculated by the multivariate analyses in this cohort) leads on average to a loss of 12 bps per year. Participants in the third and fourth quartile of urinary antimony concentration had, on average, a loss of LTL of approximately 115 and 147 bps, respectively. If these findings reflect a relationship measured without bias, then the difference between individuals of the same chronological age in the highest quartile versus the lowest quartile would be equivalent to 12.25 calendar years.

The underlying biological mechanism of how antimony may play a role in telomere shortening could potentially involve the promotion of oxidative stress and generation of reactive oxygen species (ROS). Telomeres are particularly sensitive to ROS because they are rich in guanine residues, and guanine can be oxidized to 8 hydroxyguanine, which is unstable (Mason and Perdigones 2013). Both pentavalent and trivalent antimonial compounds induce the generation of ROS in human and murine cell line (Mann et al. 2006; Mookerjee Basu et al. 2006; Rais et al. 2000; Wyllie and Fairlamb 2006).

The mechanisms of apoptosis are highly complex and sophisticated, involving an energy-dependent cascade of molecular events. There are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. Both of these pathways converge on the same terminal pathway involving cleavage of caspase-3. This results in DNA fragmentation, leading to formation of apoptotic bodies, expression of ligands for phagocytic cell receptors, and terminal uptake by phagocytic cells (Elmore 2007). Therefore, once caspase-3 is activated, there seems to be an irreversible commitment towards cell death.

Several in vitro studies have described the induction of apoptosis by trivalent antimony through activation of caspase-3 (Lecureur et al. 2002a, b; Losler et al. 2009; Mann et al. 2006; Muller et al. 1998; Wyllie and Fairlamb 2006). Lecureur et al. (2002b) pointed out that the cytotoxic potassium antimonyl tartrate concentrations action on human lymphoma Daudi cells and on B lymphocytes from leukemia patients were in micromolar concentrations; concentrations that are likely to be achievable in vivo in humans (Schulert et al. 1966).

Loss of telomere length induces the same Sb₂O₃-induced cellular apoptotic pathways. In an elegant experimental design, Bermudez et al.(2006) using normal ovarian epithelial cell lines (that are telomerase-negative) transfected with a plasmid construct containing human telomerase reverse transcriptase (hTERT) cDNA, found that expression of hTERT was sufficient and specific to reduce caspase-mediated cellular apoptosis. Cells transfected with hTERT had increased mean telomere length, increased growth capacity, and increased cell survival. Furthermore, exposure of hTERT-transfected cells to staurosporine, an inducer of apoptosis, resulted in suppressed caspase-3 activation. Conversely, cells transfected with dominant-negative (DN) hTERT had restored caspase-3 activation after staurosporine treatment, at a similar level to that found in control cells. Because antimony exposure and telomere loss induce the same pathways, and telomere loss throughout life may also be due to cumulative oxidative damage (d’Adda di Fagagna et al. 2003; von Zglinicki et al. 2005), it may be biologically plausible that antimony causes shortened LTL through the induction of cellular oxidative stress. Alternatively, antimony toxicity may accelerate apoptosis of cells already undergoing telomere loss and senescence.

After stratification by age groups, we found that there was a statistically significant association of antimony with shorter LTL in middle age and older adults groups but not in young adults. These differences may be due to the fact that older adults may undergo telomere loss due to repeated activation of immune cells – particularly CD8 T lymphocytes – from persistent viral infection, lifelong exposure to various pathogens, as well as oxidative stress (Effros 2011). Reactivation of latent viruses, such as several herpes viruses, frequently occurs in older people that are more likely to be under conditions of immunosuppression; whereas herpes viruses are infrequently re-activated in younger adults, particularly in those with normally functioning immune systems (Effros 2011). There are several reports of antimony activating latent varicella zoster virus among patients treated for leishmaniasis or schistosomiasis with antimonial compounds (Barros et al. 2014; Hartzell et al. 2006; Ritchken and Kantor 1947). Since antimony accumulates highly and rapidly in blood cells after in vivo administration (Winship 1987) and also induces oxidative damage in leukocyte cells (Muller et al. 1998; Wyllie and Fairlamb 2006), antimony exposure may contribute to cumulative oxidative damage. Therefore, it might increase cell senescence that will be manifested to a greater extent in older adults compared to younger adults.

Although the strength of our study is that it is based on a nationally representative survey, the main study limitation is the cross-sectional design of NHANES. Thus, we cannot infer a causal association. Although we entered the percentage of lymphocytes and granulocytes as separate variables in our analyses, we could not exclude the confounding effect of the proportion of different leukocyte subtypes. In younger individuals, telomeres are generally shorter in granulocytes compared to T lymphocytes; whereas, in older individuals there is a reverse, with granulocytes having longer telomeres than T lymphocytes, possibly reflecting an increase of memory T cells, which have shorter telomeres compared to naïve T lymphocytes (Weng 2001). An additional limitation is that we could not exclude the effect of co-exposure to arsenic. Antimony, which belongs to the same group (Group V) of the periodic table as arsenic, shares numerous biological features with this metalloid (Gebel 1997). Arsenic trioxide induces oxidative damage, activates caspase-3, and induces apoptosis in human leucocyte cells, and the toxic cellular effects are greater than that induced by a corresponding dose of antimonial compounds (Losler et al. 2009). In the NHANES 1999–2000 and 2001–2002 cycles, arsenic was not measured, thus we may not know whether the observed associations might be due to arsenic co-exposure.

In conclusion, in the largest study to date of the association between antimony exposure and telomere length, we found an independent association between environmental exposures to antimony and shorter LTL after adjustment for potential confounders. Further studies are needed to confirm our findings of antimony on telomere shortening and the potential role in chronic diseases involved in cellular senescence and aging.