MOLECULAR CARCINOGENESIS
Arsenic Exposure through Drinking Water Leads to Senescence and Alteration of Telomere Length in Humans: A Case-Control Study in West Bengal, India Debmita Chatterjee,1 Pritha Bhattacharjee,2 Tanmoy J. Sau,3 Jayanta K. Das,4 Nilendu Sarma,3 Apurba K. Bandyopadhyay,1 Sib Sankar Roy,5 and Ashok K. Giri1* 1
Molecular and Human Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Department of Environmental Science, University of Calcutta, Kolkata, India 3 Sir Nil Ratan Sircar Medical College and Hospital, Kolkata, India 4 Department of Dermatology, West Bank Hospital, Howrah, West Bengal, India 5 Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India 2
Arsenic (As) induces pre-malignant and malignant dermatological lesions, non-dermatological health effects and cancers in humans. Senescence involves telomere length changes and acquisition of senescence-associated secretory phenotype (SASP), which promotes carcinogenesis. Though in vitro studies have shown that As induces senescence, population based studies are lacking. We investigated the arsenic-induced senescence, telomere length alteration and its contribution towards development of As-induced skin cancer. The study participants included 60 each of As-exposed individuals with skin lesion (WSL), without skin lesions (WOSL) and 60 unexposed controls. Exposure assessment of drinking water and urine was done. SA b-gal activity, ELISA, and quantification of senescence proteins, alternative lengthening of telomere (ALT) associated proteins and telomerase activity were performed. Relative telomere length (RTL) was determined by qPCR. A significantly higher number of senescent cells, over-expression of p53 and p21 were observed in the As-exposed individuals when compared to unexposed. SASP markers, MMP-1/MMP-3 were significantly higher in the WSL but not IL-6/IL-8. A significant increase of RTL was observed in the WSL group, which was telomerase-independent but exhibited an overexpression of ALT associated proteins TRF-1 and TRF-2 with higher increase in TRF-2. An increased risk for developing Asinduced skin lesions was found for individuals having RTL greater than 0.827 (odds ratio, 13.75; 95% CI: 5.66–33.41; P < 0.0001). Arsenic induces senescence in vivo, but the SASP markers are not strictly over-expressed in the As-induced skin lesion group, whereas telomerase-independent elongation of telomere length might be useful for predicting the risk of development of As-induced skin lesions. © 2014 Wiley Periodicals, Inc. Key words: alternative lengthening; arsenic; humans; senescence; telomere INTRODUCTION In West Bengal, India, more than 26 million individuals are exposed to arsenic (As) through drinking contaminated ground water, which according to World Health Organization (WHO) should be below 10 mg/L, considered as maximum permissible limit (MPL) [1,2]. In humans, chronic exposure to arsenic has been associated with an increased cancer risk of skin, internal organs, as well as non-cancerous health outcomes [3,4]. It is interesting to note that only 15–20% of the As-exposed individuals show skin lesions including pre-malignant (hyperkeratosis) or malignant forms of skin cancer [squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and Bowen’s disease (BD)]. Studies have identified several possible modes of arsenic toxicity and carcinogenicity including oxidative stress inducing DNA damage, erythrocyte membrane deformation, inflammation, and impaired DNA repair [5–7]. Induction of senescence is associated with genotoxic insult and oxidative stress [8]. Senescence primarily, is a tumor-suppressive mechanism involving cell cycle arrest to prevent malignant transformaß 2014 WILEY PERIODICALS, INC.
tion. However, during senescence, a cell undergoes extensive changes in its protein expression and secretion profile known as the senescence associated secretory phenotype (SASP) altering the tissue microenvironment by changing senescent cells to proAbbreviations: SASP, senescence associated secretory phenotype; IL-6 and IL-8, interleukin 6 and 8; MMP-1 and MMP-3, matrix metalloproteinase-1 and 3; ALT, alternative lengthening of telomeres; APB, acute promyelocytic leukemia body; TRF, telomere repeat factor; WSL, with skin lesion; WOSL, without skin lesions; PBMC, peripheral blood mono-nuclear cells; TL, telomere length; RTL, relative telomere length; TRF, terminal restriction fragment; OR, odds ratio. The authors declare they have no actual or potential competing financial interests. Grant sponsor: Indian Council of Medical Research (ICMR) Under Indo-US Collaborative Program; Grant number: 65/15/2008-NCD-I; Grant sponsor: Council of Scientific and Industrial Research (CSIR) Emeritus Project; Grant number: 21(0885)/12/EMR-II to AKG *Correspondence to: Emeritus Scientist (CSIR), Molecular and Human Genetics Division, CSIR-Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India. Received 9 October 2013; Revised 14 February 2014; Accepted 25 February 2014 DOI 10.1002/mc.22150 Published online in Wiley Online Library (wileyonlinelibrary.com).
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inflammatory cells leading to tumor promotion [8]. Hence senescence acts as a “double-edged sword” being a precursor of cancer in both cancer and normal cells. There are two major signaling pathways identified leading to senescence; one is the p53 dependent pathway through p21 and the other is the p53 independent p16/pRb pathway [9]. The pro-tumorigenic SASP factors consist of interleukins, chemokines, secreted proteases and secreted insoluble proteins. Interleukin 6 (IL-6) and interleukin 8 (IL8) are the most prominent soluble factors while the matrix metalloproteases MMP-1 and MMP-3 are the most noticeable among the secreted proteases [8]. The hallmark of senescent cell detection is the senescenceassociated b-galactosidase (SA-b-gal) activity [10], which reflects the increase in lysosomal enzymatic activity at pH 6.0. Telomere length alteration is an important feature of senescence [11]. Alteration in the telomere structure can lead to multiple abnormalities like chromosomal instability, tumorigenesis and cancer [12]. In vitro experiments with arsenic have shown contrasting effects like lengthening as well as shortening of telomere in a dose-dependent manner [13]. A number of human cancers including skin cancer have been associated with longer telomere length [14,15]. Telomerase overexpression induced increase in telomere length has been reported in more than 85% of human cancers [16]. Telomere length maintenance can also occur by a telomerase-independent mechanism identified as alternative lengthening of telomeres or ALT [17]. Cells, using the ALT mechanism have unique characteristics like heterogenous telomere length, nuclear structures known as ALT associated PML bodies (APB’s) and extra-chromosomal telomeric DNA [18]. These APB’s contain telomere specific proteins like Telomere Repeat Factor 1 (TRF1), Telomere Repeat Factor 2 (TRF2), Protection of Telomeres 1 (POT1) which help in recombination mediated telomere lengthening [18]. TRF1 and TRF2 have been up-regulated in several cancers and are crucial for malignant transformation [19–21]. The mechanism of As-induced carcinogenesis is still not clear. The objective of the present study is to evaluate the complex interaction among cellular senescence, SASP markers and telomeric regulation in individuals susceptible toward pre-malignant or malignant forms of As-induced dermatological lesions.
much higher (30–620 mg/L) compared to the recommended MPL of 10 mg/L [2,22]. The arsenic unexposed controls were recruited from East Midnapore district, where arsenic content was within the safe limit laid down by WHO [2]. The detailed procedure of field survey, strategy for genetically unrelated case-control selection, and sample collection have been described in our previous studies [3,4]. Demographic details of the recruited individuals, who had been residing in the area for at least 10 years, were recorded. Expert dermatologists screened individuals with As-specific skin lesions comprising of hyperkeratosis, SCC, BCC, and BD. Sixty individuals matched for age, sex, tobacco-usage, and socio-economic status, were recruited for each of the three study groups [Asexposed with skin lesion (WSL); As-exposed without skin lesions (WOSL); Unexposed controls]. After a well informed, written consent from the study participants, blood, urine, and drinking water samples were collected for further analysis. The study was in accordance to the Declaration of Helsinki II and approved by the Institutional Ethical Committee on human subjects of CSIR-Indian Institute of Chemical Biology. Sample Collection and Exposure Analysis Urine and drinking water samples were collected, stored in 208C prior analysis and assessed for total As-content as discussed earlier [4,23]. As-measurement was performed by flow injection-hydride generation-atomic absorption spectrometry (FI-HGAAS) using a Model Analyst-700 spectrometer of Perkin Elmer (Waltham, MA) at CSIR-Indian Institute of Chemical Biology. Isolation of Serum, Peripheral Blood Mononuclear Cells (PBMC), DNA and Proteins Serum was isolated by centrifugation of clotted blood at 2000 rpm at 48C. Peripheral blood mononuclear cells (PBMCs) were obtained from blood (in EDTA vacutainers) by density gradient centrifugation using HistopaqueTM (Sigma, GmBH, Germany). Cell viability was determined by trypan-blue dye exclusion and was always greater than 95% [24]. DNA was isolated from whole blood using QIAamp1 DNA Mini Kit (Qiagen, GmBH, Germany). The protein was isolated from the PBMC using AllPrep1 RNA/Protein Isolation kit, Qiagen. Cellular Senescence Assay
MATERIALS AND METHODS Study Sites and Subject Selection The arsenic-exposed study participants were selected from the highly arsenic affected districts of West Bengal namely Murshidabad, North 24-Parganas, and Nadia, where arsenic content in drinking water was
Molecular Carcinogenesis
PBMC of the study groups were processed according to the Cellular Senescence Assay kit following manufacturers’ protocol (Chemicon International, Billerica, MA). The number of SA-b-gal positive cells (blue) were determined by counting 300 cells from randomly chosen fields under low-power (100) and expressed as a percentage of all counted cells.
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Measurement of SASP Markers (IL-6, IL-8, MMP-1, and MMP-3) IL-6, IL-8, MMP-1, and MMP-3 concentrations of serum samples in each of the three groups were measured using IL-6 and IL-8 ELISA kits (Thermo Scientific, Rockford, IL), and MMP-1 and MMP-3 kit (RayBiotech, Inc., Norcross, GA) according to manufacturer’s instructions. Telomere Length Assessment Telomere length (TL) was measured using relative telomere length (RTL) measurement using qPCR from whole blood genomic DNA in each of the study groups. The primers and PCR conditions were adapted from Cawthon [25], to measure TL. Briefly, 20 ng of DNA was prepared for each sample to measure the RTL and concentrations for telomere and albumin primers were 900 nM each. Final reaction volume was 20 ml per reaction containing 10 ml of Brilliant SYBR Green QPCR master mix (Agilent Technologies, Santa Clara, CA). Determination of RTL by this method was based on the ratio of telomere repeat copy number (T) to single copy gene copy number (S) expressed as T/S ratio. All experiments were done in triplicate and conducted in Mx3000P (Stratagen, Agilent Technologies, Santa Clara, CA). To further validate our observation, the absolute TL was measured using Southern blot. A subset of randomly selected 25 samples used earlier for qPCR, from each of the three study groups were matched and used for TL measurement. Purified gDNA (2 mg) was taken to determine the absolute TL using terminal restriction fragment (TRF) using Telomere Length Assay Kit (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturer’s protocol. Density measurements were performed with the Image J software (NIH, Bethesda, MD). TRF Plength was calculated using the formula TRF ¼ (ODi)/ P (ODi/Li) where ODi is the chemiluminescent signal and Li is the length of the TRF at position i. Western Blotting PBMC-isolated proteins were used for Western blotting, as described previously [23]. Primary antibodies were: mouse monoclonal p16, mouse monoclonal anti-TRF2 (Calbiochem, Temecula, CA); rabbit polyclonal IgG p21, rabbit polyclonal IgG p53, mouse monoclonal b-actin (Santa Cruz Biotechnology Inc., Dallas, Texas); mouse monoclonal anti-TRF1 (Millipore, Temecula, CA). Secondary antibody (alkaline phosphatase conjugated), purchased from Calbiochem, was diluted (1:2000 in 1X TBST) and hence detected with NBT-BCIP substrate (Calbiochem). Densitometric analysis was performed by Image J software (NIH). The fold change is expressed after normalization with the loading control (b-actin). Telomerase Activity Assay Isolated PBMC (2 105 cells) from a subset of 20 randomly chosen (age–sex matched) samples used Molecular Carcinogenesis
earlier for qPCR, in each of the three study groups, were subjected to lysis for protein extraction using the Telo TAGGG Telomerase PCR ELISA kit (Roche Applied Science), following the manufacturer’s protocol. A sample is considered to be telomerase positive if the difference in absorbance of the samples and the negative control (degraded telomerase with RNase treatment) is higher than the kit recommended 0.2. The absorbance of the supplied positive control (a known telomerase positive cell line extract) should be 1.5 A450–A690 and was 2.5 in our case. Statistical Analysis The statistical analysis was done using GraphPad InStat Software (San diego, CA). Since this is a matched case-control study pairwise t-test was performed to compare the central tendencies of continuous independent variables (like age, As-content in water, urine) between the WSL, WOSL, and unexposed groups. We used logistic regression (XLSTAT Version 2013.4.05) to determine the telomere length (RTL0.5) that corresponds to a probability of 0.5 of having arsenic specific skin lesion (Figure S1). This RTL of 0.827 denoted henceforth as RTL0.5 was used as the threshold value for predicting the risk of development of skin lesion in the study participants, as measured by odds ratio (OR) where the As-exposed individuals without skin lesions were taken as referent. RESULTS Demographic Characteristics and Arsenic Exposure of the Participants The detailed demographic characteristics of the study population are summarized in Table 1. The Ascontent in the drinking water and urine were significantly higher (P < 0.001) when compared to unexposed individuals. Arsenic Exposure Induces Senescence Via the p53-p21 Pathway On measuring the extent of As-induced senescence we found that the percentage of senescent cells (Figure 1a–c) was significantly higher (P < 0.001) in the As-exposed population (8.5 1.04%) than the unexposed (2.3 0.59%). Quantitative analysis of the senescence-inducer proteins revealed that p16 was about two-fold higher in the unexposed controls than the exposed group whereas p21 and p53 was about 2.5 and 1.46-fold, respectively, higher in the exposed group than the unexposed controls (Figure 1d and e). Differential Expression of SASP Markers With the Presence of Arsenic-Induced Skin Lesions The expression of SASP markers showed that cytokines IL-6 (Figure 2a) and IL-8 (Figure 2b) was significantly higher (P < 0.001) in the WOSL group when compared to the WSL and unexposed group.
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Table 1. Demographic Characteristic of the Study Participants
Parameters Total subjects (n) Age (in years; mean SD) Gender [n (%)] Male Female Tobacco usage [n (%)] Smoker Nonsmoker Occupation [n (%)] Farmer Business Teacher Service Student Unemployed Housewife Arsenic content in mg/L [mean SD] Drinking water Urine
Arsenic-unexposed group N (%)
Arsenic-exposed without skin lesion group N (%)
Arsenic-exposed with skin lesion group N (%)
60 44.63 11.41
60 44.85 11.55
60 44.74 11.21
32 (53.33) 28 (46.67)
35 (58.33) 25 (41.67)
37 (61.67) 23 (38.33)
14 (23.33) 46 (76.67)
10 (16.67) 50 (83.33)
11 (18.33) 49 (81.67)
32 (53.33) 2 (3.33) 1 (1.67) 3 (1.67) 5 (8.33) 2 (3.33) 15 (25)
35 (58.33) 1 (1.67) 0 2 (3.33) 4 (6.67) 3 (5.0) 14 (23.33)
34 (56.67) 1 (1.67) 2 (3.33) 1 (1.67) 3 (5.0) 3 (5.0) 16 (26.67)
9.20 0.31 30.5 0.51
143 59.0a 318 95.7a
153 65.5a 290 83.4a
Paired t-test, Pa < 0.001 (two tailed); when compared to unexposed controls.
MMP-1 (Figure 2c) and MMP-3 (Figure 2d) levels were found to be significantly higher (P < 0.001) in the WSL group when compared to the WOSL and unexposed controls. MMP-1 and MMP-3 were also
significantly higher (P < 0.01 and P < 0.001, respectively) in the WOSL group when compared to the unexposed. The concentrations of the SASP markers are listed in .
Figure 1. Comparison of arsenic-induced senescence in the study population. (a) Arsenic exposed population shows higher presence of SA b-gal positive (blue cells) in comparison to (b) Unexposed population. (c) Graph showing the percentage of SA b-gal positive cells upon arsenic-exposure in comparison to the unexposed population. (d) Protein expression for p16, p21, p53, and b-actin from PBMC of unexposed controls, WOSL and WSL. (e) Quantitative analysis of senescence inducer proteins p16, p21 and p53 from PBMC. P < 0.01; P < 0.001[Paired t-test (Wilcoxon matched pairs test) for SA b-gal and One way ANOVA for Western blot].
Molecular Carcinogenesis
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Figure 2. Measurement of SASP factors. Expression of (a) serum IL-6 and (b) IL 8 and extracellular proteases (c) MMP-1, and (d) MMP-3. P < 0.01; P < 0.001 (One way ANOVA).
Telomere Length Alteration Is Associated With ArsenicInduced Skin Lesion Telomere length status exhibited a negative correlation with age for all the study groups (Figure 3a). The distribution of TL with age in the three study groups is shown in Figure 3b. A significant increase (P < 0.001) in TL was observed in the WSL individuals (0.899 0.084) when compared to unexposed controls (0.749 0.044) as well as WOSL individuals (0.767 0.0728). Similar results were observed when absolute TL was compared between the study groups (Table S2). However, no significant increase in TL was found in the WOSL individuals with that of the unexposed controls (Figure 3c). WSL individuals had significantly higher TL for all the age groups (Figure 3d). RTL of the WSL group was positively associated with the As-content in drinking water and urine having an R2 value of 0.890 and 0.954, respectively (Figure 3e). We found that RTL value greater than RTL0.5 was significantly associated (P < 0.0001) with an increased risk (OR: 13.75; 95% CI: 5.66–33.41) towards development of As-induced skin lesion. To know the mechanism behind the differential TL, telomerase activity was measured in the three study groups. All the study groups, unexposed (0.119 0.04), Molecular Carcinogenesis
WOSL group (0.095 0.04) and WSL (0.121 0.05) had telomerase activity less than the kit recommended threshold of 0.2 and hence none of them were considered as telomerase positive (Figure 4a). TRF1 expression was higher (P < 0.01) in the WSL individuals by about 1.3 and 1.5-fold when compared with the unexposed as well as the WOSL group. Similarly, TRF2 expression was about 4 and 4.4-fold higher (P < 0.001) in WSL when compared with the unexposed and the WOSL group respectively (Figure 4b and c). DISCUSSION Earlier reports on As-induced senescence in vitro [26,27], prompted us to carry out a population based study on the alteration of senescence and telomeric regulation using blood based biomarkers. Earlier, studies have reported that the gene expression signatures present in the peripheral blood is consistent with most of the tissue types within the body like liver, heart, kidney, lung, etc. including dermatological lesion tissues in humans [28,29]. It is also well known that telomeres and telomerase play a major role at the aging-cancer interface [9]. To the best of our knowledge, this is the first study to look into As-induced senescence in human population and
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Figure 3. Measurement of telomere length. (a) Correlation between telomere length of genomic DNA from whole blood between unexposed controls, WOSL and WSL with age using Pearson's correlation coefficient (r). (b) Distribution of telomere length with respect to age in the study groups. (c) RTL comparison in the study groups. (d) Measurement of RTL with respect to age-subgroups in the study population. (e) Correlation of RTL with arsenic content in water and urine; R2 ¼ 0.890 (for water) and R2 ¼ 0.954 (for urine). P < 0.01; P < 0.001 (One way ANOVA).
telomere length alteration, on the basis of As-induced skin lesions. Our results show that the As-exposed population, irrespective of skin lesion status had significantly Molecular Carcinogenesis
increased senescent cells than the unexposed population. Arsenic might act as a xenobiotic factor, inducing senescence. The levels of the senescence inducer proteins like p16, p21, and p53 in the WSL
ARSENIC CAUSES TELOMERIC ELONGATION
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Figure 4. Mechanism of telomere length elongation upon arsenic exposure. (a) Telomerase activity measurement from PBMC isolated protein of Unexposed, WOSL and WSL groups (t-test, P > 0.05). (b) Protein expression for TRF1, TRF2, and b-actin from lymphocyte of unexposed controls, WOSL, WSL. (c) Densitometric analysis of the telomeric proteins TRF1 and TRF2. P < 0.01; P < 0.001 (One way ANOVA).
and WOSL group shows up-regulation of the p53 pathway involving p53 and its downstream effector p21 in the exposed group but not p16. Similar results were also reported earlier by other authors involving in vitro studies with As-treatment [26]. In order to understand whether the hyper-secretory factors of senescence are playing a causal role in Asinduced carcinogenesis we assessed the expression of SASP markers [8]. We observed that there is a significant increase in the IL-6 and IL-8 levels in the WOSL group whereas the invasion markers MMP-1 and MMP-3 are significantly higher in the WSL group. In a study by Ahmed et al., the IL-8 concentration in the human cord blood plasma showed a U-shaped association, increasing consistently in the group with urinary As-content 71–211 mg/L and even higher in the group with urinary-As greater than 211 mg/L. This is in accordance with our study where the urinary-As concentration were greater than 211 mg/L [30]. Thus we can infer that although arsenic is inducing the SASP, it is not associated with As-induced skin lesions and is not the pivotal factor contributing to the development of As-induced carcinogenesis. Our results on telomere length were consistent with the already established fact that telomeres act as a biomarker for replicative aging [31], evident from the negative correlation with age. On comparing the telomere length in our three study groups we observed a strikingly significant increase in telomere Molecular Carcinogenesis
length in the WSL group when compared to WOSL and unexposed group. Telomere length increase in the WSL individuals was consistent for all the age groups. This might be an indication that arsenic has a steady effect on telomere length, independent of age. In our study, individuals having RTL greater than RTL0.5 showed a significantly increased risk of developing skin lesions with an OR: 13.75 (95% CI, 5.66–33.41), when compared with the referent. Hence, it is noteworthy, that RTL could be used for predicting the risk of As-induced skin lesions in humans. The TL in the WSL group was positively correlated with As-content in water and urine, having better correlation with urinary-As content. UrinaryAs being the best measure of recent As-exposure supports our observation [32]. Previous studies have reported that treatment of human cells with arsenic resulted in maintenance of TL [33]. Recently, a population based study, conducted on women, revealed a positive association between TL and Asexposure [34]. Depending on our results, we may hypothesize that the increase in TL in the As-induced skin lesion group may be the key factor for predisposition of this group towards development of precancerous or cancerous skin lesions. To gain a mechanistic insight into the TL maintenance in WSL group we performed the TRAP assay from peripheral blood cells and found that TL elongation was telomerase-independent. Hence, our
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observations indicate that the ALT pathway may play a role in telomeric elongation in As-exposed skin lesion cases. Since our population is chronically exposed to arsenic for at least 10 years, it is expected that the lengthening of telomeres resulted from the long-term exposure of arsenic through drinking water. The APB associated shelterin complex proteins TRF1 and TRF2 is known to mediate telomere lengthening by ALT [18,35]. The proteins involved in ALT that is, TRF1 and TRF2 were found to be significantly higher in the WSL group when compared with the WOSL as well as unexposed controls. However, no significant difference was observed between the WOSL and the unexposed group indicating that the telomeric proteins are being up-regulated in the WSL group only. Our observations are in full agreement with the previous in vitro work [36], where As-treated human gastric cancer cells showed upregulation of TRF1 and TRF2 without telomere shortening, hence contradicting earlier reports [37]. TRF1 and TRF2 proteins have been identified as essential components of APB’s whereby depletion of these proteins directly inhibited the formation of APB’s. Since, the inhibition of APB’s is reflected in the impairment of the ALT-mediated telomere mainte-
nance, levels of TRF1 and TRF2 needs to be maintained for ALT [38]. Also recently, TRF2 has been associated with telomere maintenance [39]. Senescence is associated with APB formation and earlier studies have reported increased APB formation with p21 over-expression [40]. DNA compaction renders the recombination mediated ALT inactive. However, sodium arsenite has been shown to induce histone hyper-acetylation leading to an open chromatin state [41]. It can be hypothesized that arsenic leads to increased accessibility of DNA towards the APB associated proteins like TRF1 and TRF2, promoting the ALT mediated TL elongation. In summary, our findings indicate that As-exposure induces senescence in vivo, occurring via the p53 dependent pathway, inducing downstream p21, leading to the SASP markers being elevated upon As-exposure irrespective of the skin lesion status. However interestingly, TL elongation is found to be strictly associated with the occurrence of As-induced skin lesion (Table 2). Telomere length may be used for predicting the risk of susceptibility of an individual towards development As-induced dermatological lesions. We found that the telomerase-independent telomere elongation is associated with up-regulation
Table 2. Summary of Arsenic Exposure Associated Effects in the Study Groups Parameters studied
Source material
WOSL
WSL
WOSL/WSL
PBMC
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p16 level
PBMC
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p21 level
PBMC
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P53 level
PBMC
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—
IL-6
Serum
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IL-8
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MMP-1
Serum
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MMP-3
Serum
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Whole blood
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PBMC
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SA-b gal positive cells
Telomere length Telomerase activity
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" or #: Significant change (either increase/decrease) in the WOSL or WSL group with respect to unexposed (P < 0.05). or
: Significant change (either increase/decrease) in the WOSL with respect to WSL group (P < 0.05).
—, No significant change; Down-ward arrow, decrease; Up-ward arrow, increase. Molecular Carcinogenesis
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of telomeric proteins suggesting that recombinationmediated ALTs could be the mechanism behind Asinduced tumorigenesis. Further studies are required to understand this molecular mechanism, specifically from As-induced skin cancer tissues as well as other cancer types. In due course, this may help in molecular diagnosis to detect As-toxicity. ACKNOWLEDGMENTS This work was supported by Indian Council of Medical Research (ICMR) under Indo-US collaborative program [grant number 65/15/2008-NCD-I]; and Council of Scientific and Industrial Research (CSIR) Emeritus Project [grant number 21(0885)/12/EMR-II to A.K.G.]. We are indebted to Mr. Somnath Paul, Junior Research Fellow at CSIR-Indian Institute of Chemical Biology for his helpful discussions and immense support throughout the work. We are grateful to Mr. Jayanta Das, support staff at CSIRIndian Institute of Chemical Biology for on-field collection of the precious human samples. REFERENCES 1. Mondal D, Banerjee M, Kundu M, et al. Comparison of drinking water, raw rice and cooking of rice as arsenic exposure routes in three contrasting areas of West Bengal, India. Environ Geochem Health 2010;32:463–477. 2. Guidelines for Drinking Water Quality. Health criteria and other supporting information 2. Geneva: World Health Organisation; 1996. pp. 940–949. 3. Ghosh P, Banerjee M, De Chaudhuri S, et al. Comparison of health effects between individuals with and without skin lesions in the population exposed to arsenic through drinking water in West Bengal, India. J Expo Sci Environ Epidemiol 2007;17:215–223. 4. Paul S, Das N, Bhattacharjee P, et al. Arsenic-induced toxicity and carcinogenicity: A two-wave cross-sectional study in arsenicosis individuals in West Bengal, India. J Expo Sci Environ Epidemiol 2013;23:156–162. 5. Biswas D, Banerjee M, Sen G, et al. Mechanism of erythrocyte death in human population exposed to arsenic through drinking water. Toxicol Appl Pharmacol 2008;230:57–66. 6. Jasso-Pineda Y, Díaz-Barriga F, Calderón J, et al. DNA damage and decreased DNA repair in peripheral blood mononuclear cells in individuals exposed to arsenic and lead in a mining site. Biol Trace Elem Res 2012;146:141–149. 7. Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem 2004;266:37–56. 8. Copp e JP, Desprez PY, Krtolica A, Campisi J. The senescenceassociated secretory phenotype: The dark side of tumor suppression. Annu Rev Pathol 2010;5:99–118. 9. Campisi J, Kim SH, Lim CS, Rubio M. Cellular senescence, cancer and aging: The telomere connection. Exp Gerontol 2001;36:1619–1637. 10. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 1995;92:9363–9367. 11. Karlseder J, Smogorzewska A, de Lange T. Senescence induced by altered telomere state, not telomere loss. Science 2002;295:2446–2449. 12. Calado RT, Cooper JN, Padilla-Nash HM, et al. Short telomeres result in chromosomal instability in hematopoietic cells and precede malignant evolution in human aplastic anemia. Leukemia 2012;26:700–707.
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13. Zhang TC, Schmitt MT, Mumford JL, et al. Effects of arsenic on telomerase and telomeres in relation to cell proliferation and apoptosis in human keratinocytes and leukemia cells in vitro. Carcinogenesis 2003;24:1811–1817. 14. Gramatges MM, Telli ML, Balise R, Ford JM. Longer relative telomere length in blood from women with sporadic and familial breast cancer compared with healthy controls. Cancer Epidemiol Biomarkers Prev 2010;19:605–613. 15. Anic GM, Sondak VK, Messina JL. Telomere length and risk of melanoma, squamous cell carcinoma, and basal cell carcinoma. Cancer Epidemiol 2013;37:434–439. 16. Neumann AA, Reddel RR. Telomere maintenance and cancer—Look, no telomerase. Nat Rev Cancer 2002;2:879– 884. 17. Nabetani A, Ishikawa F. Alternative lengthening of telomeres pathway: Recombination-mediated telomere maintenance mechanism in human cells. J Biochem 2011;149:5–14. 18. Bhattacharyya S, Sandy A, Groden J. Unwinding protein complexes in ALTernative telomere maintenance. J Cell Biochem 2010;109:7–15. 19. Lantuejoul S, Raynaud C, Salameire D, et al. Telomere maintenance and DNA damage responses during lung carcinogenesis. Clin Cancer Res 2010;16:2979–2988. 20. Dong W, Shen R, Wang Q, et al. Sp1 upregulates expression of TRF2 and TRF2 inhibition reduces tumorigenesis in human colorectal carcinoma cells. Cancer Biol Ther 2009;8:2166– 2174. 21. Biroccio A, Rizzo A, Elli R, et al. TRF2 inhibition triggers apoptosis and reduces tumourigenicity of human melanoma cells. Eur J Cancer 2006;42:1881–1888. 22. West Bengal Public Health Engineering Department (WBPHED). http://www.wbphed.gov.in (April 2012, last updated, October 2012, last accessed). 23. Banerjee N, Paul S, Sau TJ, et al. Epigenetic modifications of DAPK and p16 genes contribute to arsenic-induced skin lesions and nondermatological health effects. Toxicol Sci 2013;135: 300–308. 24. Kuwana T, Mackey MR, Perkins G, et al. Bid, Bax and lipid cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 2002;111:331–342. 25. Cawthon RM. Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Res 2009;37:e21. 26. Cheng H, Qiu L, Zhang H, et al. Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells. Acta Biochim Biophys Sin 2011;43:204–209. 27. Martín-Pardillos A, Sosa C, Sorribas V. Arsenic increases Pimediated vascular calcification and induces premature senescence in vascular smooth muscle cells. Toxicol Sci 2013;131: 641–653. 28. Liew CC, Ma J, Tang HC, Zheng R, Dempsey AA. The peripheral blood transcriptome dynamically reflects system wide biology: A potential diagnostic tool. J Lab Clin Med 2006; 147:126–132. 29. Matsushita T, Hasegawa M, Hamaguchi Y, Takehara K, Sato S. Longitudinal analysis of serum cytokine concentrations in systemic sclerosis: Association of interleukin 12 elevation with spontaneous regression of skin sclerosis. J Rheumatol 2006;33: 275–284. 30. Ahmed S, Mahabbat-e Khoda S, Rekha RS, et al. Arsenicassociated oxidative stress, inflammation, and immune disruption in human placenta and cord blood. Environ Health Perspect 2011;119:258–264. 31. Monickaraj F, Aravind S, Gokulakrishnan K, et al. Accelerated aging as evidenced by increased telomere shortening and mitochondrial DNA depletion in patients with type 2 diabetes. Mol Cell Biochem 2012;365:343–350. 32. Buchet JP, Lauwerys R, Roels H. Comparison of the urinary excretion of arsenic metabolites after a single dose of sodium
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SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Arsenic Exposure through Drinking Water Leads to Senescence and Alteration of Telomere Length in Humans: A Case-Control Study in West Bengal, India Debmita Chatterjee,1 Pritha Bhattacharjee,2 Tanmoy J. Sau,3 Jayanta K. Das,4 Nilendu Sarma,3 Apurba K. Bandyopadhyay,1 Sib Sankar Roy,5 and Ashok K. Giri1* 1
Molecular and Human Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India Department of Environmental Science, University of Calcutta, Kolkata, India 3 Sir Nil Ratan Sircar Medical College and Hospital, Kolkata, India 4 Department of Dermatology, West Bank Hospital, Howrah, West Bengal, India 5 Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India 2
Arsenic (As) induces pre-malignant and malignant dermatological lesions, non-dermatological health effects and cancers in humans. Senescence involves telomere length changes and acquisition of senescence-associated secretory phenotype (SASP), which promotes carcinogenesis. Though in vitro studies have shown that As induces senescence, population based studies are lacking. We investigated the arsenic-induced senescence, telomere length alteration and its contribution towards development of As-induced skin cancer. The study participants included 60 each of As-exposed individuals with skin lesion (WSL), without skin lesions (WOSL) and 60 unexposed controls. Exposure assessment of drinking water and urine was done. SA b-gal activity, ELISA, and quantification of senescence proteins, alternative lengthening of telomere (ALT) associated proteins and telomerase activity were performed. Relative telomere length (RTL) was determined by qPCR. A significantly higher number of senescent cells, over-expression of p53 and p21 were observed in the As-exposed individuals when compared to unexposed. SASP markers, MMP-1/MMP-3 were significantly higher in the WSL but not IL-6/IL-8. A significant increase of RTL was observed in the WSL group, which was telomerase-independent but exhibited an overexpression of ALT associated proteins TRF-1 and TRF-2 with higher increase in TRF-2. An increased risk for developing Asinduced skin lesions was found for individuals having RTL greater than 0.827 (odds ratio, 13.75; 95% CI: 5.66–33.41; P < 0.0001). Arsenic induces senescence in vivo, but the SASP markers are not strictly over-expressed in the As-induced skin lesion group, whereas telomerase-independent elongation of telomere length might be useful for predicting the risk of development of As-induced skin lesions. © 2014 Wiley Periodicals, Inc. Key words: alternative lengthening; arsenic; humans; senescence; telomere INTRODUCTION In West Bengal, India, more than 26 million individuals are exposed to arsenic (As) through drinking contaminated ground water, which according to World Health Organization (WHO) should be below 10 mg/L, considered as maximum permissible limit (MPL) [1,2]. In humans, chronic exposure to arsenic has been associated with an increased cancer risk of skin, internal organs, as well as non-cancerous health outcomes [3,4]. It is interesting to note that only 15–20% of the As-exposed individuals show skin lesions including pre-malignant (hyperkeratosis) or malignant forms of skin cancer [squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and Bowen’s disease (BD)]. Studies have identified several possible modes of arsenic toxicity and carcinogenicity including oxidative stress inducing DNA damage, erythrocyte membrane deformation, inflammation, and impaired DNA repair [5–7]. Induction of senescence is associated with genotoxic insult and oxidative stress [8]. Senescence primarily, is a tumor-suppressive mechanism involving cell cycle arrest to prevent malignant transformaß 2014 WILEY PERIODICALS, INC.
tion. However, during senescence, a cell undergoes extensive changes in its protein expression and secretion profile known as the senescence associated secretory phenotype (SASP) altering the tissue microenvironment by changing senescent cells to proAbbreviations: SASP, senescence associated secretory phenotype; IL-6 and IL-8, interleukin 6 and 8; MMP-1 and MMP-3, matrix metalloproteinase-1 and 3; ALT, alternative lengthening of telomeres; APB, acute promyelocytic leukemia body; TRF, telomere repeat factor; WSL, with skin lesion; WOSL, without skin lesions; PBMC, peripheral blood mono-nuclear cells; TL, telomere length; RTL, relative telomere length; TRF, terminal restriction fragment; OR, odds ratio. The authors declare they have no actual or potential competing financial interests. Grant sponsor: Indian Council of Medical Research (ICMR) Under Indo-US Collaborative Program; Grant number: 65/15/2008-NCD-I; Grant sponsor: Council of Scientific and Industrial Research (CSIR) Emeritus Project; Grant number: 21(0885)/12/EMR-II to AKG *Correspondence to: Emeritus Scientist (CSIR), Molecular and Human Genetics Division, CSIR-Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India. Received 9 October 2013; Revised 14 February 2014; Accepted 25 February 2014 DOI 10.1002/mc.22150 Published online in Wiley Online Library (wileyonlinelibrary.com).
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inflammatory cells leading to tumor promotion [8]. Hence senescence acts as a “double-edged sword” being a precursor of cancer in both cancer and normal cells. There are two major signaling pathways identified leading to senescence; one is the p53 dependent pathway through p21 and the other is the p53 independent p16/pRb pathway [9]. The pro-tumorigenic SASP factors consist of interleukins, chemokines, secreted proteases and secreted insoluble proteins. Interleukin 6 (IL-6) and interleukin 8 (IL8) are the most prominent soluble factors while the matrix metalloproteases MMP-1 and MMP-3 are the most noticeable among the secreted proteases [8]. The hallmark of senescent cell detection is the senescenceassociated b-galactosidase (SA-b-gal) activity [10], which reflects the increase in lysosomal enzymatic activity at pH 6.0. Telomere length alteration is an important feature of senescence [11]. Alteration in the telomere structure can lead to multiple abnormalities like chromosomal instability, tumorigenesis and cancer [12]. In vitro experiments with arsenic have shown contrasting effects like lengthening as well as shortening of telomere in a dose-dependent manner [13]. A number of human cancers including skin cancer have been associated with longer telomere length [14,15]. Telomerase overexpression induced increase in telomere length has been reported in more than 85% of human cancers [16]. Telomere length maintenance can also occur by a telomerase-independent mechanism identified as alternative lengthening of telomeres or ALT [17]. Cells, using the ALT mechanism have unique characteristics like heterogenous telomere length, nuclear structures known as ALT associated PML bodies (APB’s) and extra-chromosomal telomeric DNA [18]. These APB’s contain telomere specific proteins like Telomere Repeat Factor 1 (TRF1), Telomere Repeat Factor 2 (TRF2), Protection of Telomeres 1 (POT1) which help in recombination mediated telomere lengthening [18]. TRF1 and TRF2 have been up-regulated in several cancers and are crucial for malignant transformation [19–21]. The mechanism of As-induced carcinogenesis is still not clear. The objective of the present study is to evaluate the complex interaction among cellular senescence, SASP markers and telomeric regulation in individuals susceptible toward pre-malignant or malignant forms of As-induced dermatological lesions.
much higher (30–620 mg/L) compared to the recommended MPL of 10 mg/L [2,22]. The arsenic unexposed controls were recruited from East Midnapore district, where arsenic content was within the safe limit laid down by WHO [2]. The detailed procedure of field survey, strategy for genetically unrelated case-control selection, and sample collection have been described in our previous studies [3,4]. Demographic details of the recruited individuals, who had been residing in the area for at least 10 years, were recorded. Expert dermatologists screened individuals with As-specific skin lesions comprising of hyperkeratosis, SCC, BCC, and BD. Sixty individuals matched for age, sex, tobacco-usage, and socio-economic status, were recruited for each of the three study groups [Asexposed with skin lesion (WSL); As-exposed without skin lesions (WOSL); Unexposed controls]. After a well informed, written consent from the study participants, blood, urine, and drinking water samples were collected for further analysis. The study was in accordance to the Declaration of Helsinki II and approved by the Institutional Ethical Committee on human subjects of CSIR-Indian Institute of Chemical Biology. Sample Collection and Exposure Analysis Urine and drinking water samples were collected, stored in 208C prior analysis and assessed for total As-content as discussed earlier [4,23]. As-measurement was performed by flow injection-hydride generation-atomic absorption spectrometry (FI-HGAAS) using a Model Analyst-700 spectrometer of Perkin Elmer (Waltham, MA) at CSIR-Indian Institute of Chemical Biology. Isolation of Serum, Peripheral Blood Mononuclear Cells (PBMC), DNA and Proteins Serum was isolated by centrifugation of clotted blood at 2000 rpm at 48C. Peripheral blood mononuclear cells (PBMCs) were obtained from blood (in EDTA vacutainers) by density gradient centrifugation using HistopaqueTM (Sigma, GmBH, Germany). Cell viability was determined by trypan-blue dye exclusion and was always greater than 95% [24]. DNA was isolated from whole blood using QIAamp1 DNA Mini Kit (Qiagen, GmBH, Germany). The protein was isolated from the PBMC using AllPrep1 RNA/Protein Isolation kit, Qiagen. Cellular Senescence Assay
MATERIALS AND METHODS Study Sites and Subject Selection The arsenic-exposed study participants were selected from the highly arsenic affected districts of West Bengal namely Murshidabad, North 24-Parganas, and Nadia, where arsenic content in drinking water was
Molecular Carcinogenesis
PBMC of the study groups were processed according to the Cellular Senescence Assay kit following manufacturers’ protocol (Chemicon International, Billerica, MA). The number of SA-b-gal positive cells (blue) were determined by counting 300 cells from randomly chosen fields under low-power (100) and expressed as a percentage of all counted cells.
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Measurement of SASP Markers (IL-6, IL-8, MMP-1, and MMP-3) IL-6, IL-8, MMP-1, and MMP-3 concentrations of serum samples in each of the three groups were measured using IL-6 and IL-8 ELISA kits (Thermo Scientific, Rockford, IL), and MMP-1 and MMP-3 kit (RayBiotech, Inc., Norcross, GA) according to manufacturer’s instructions. Telomere Length Assessment Telomere length (TL) was measured using relative telomere length (RTL) measurement using qPCR from whole blood genomic DNA in each of the study groups. The primers and PCR conditions were adapted from Cawthon [25], to measure TL. Briefly, 20 ng of DNA was prepared for each sample to measure the RTL and concentrations for telomere and albumin primers were 900 nM each. Final reaction volume was 20 ml per reaction containing 10 ml of Brilliant SYBR Green QPCR master mix (Agilent Technologies, Santa Clara, CA). Determination of RTL by this method was based on the ratio of telomere repeat copy number (T) to single copy gene copy number (S) expressed as T/S ratio. All experiments were done in triplicate and conducted in Mx3000P (Stratagen, Agilent Technologies, Santa Clara, CA). To further validate our observation, the absolute TL was measured using Southern blot. A subset of randomly selected 25 samples used earlier for qPCR, from each of the three study groups were matched and used for TL measurement. Purified gDNA (2 mg) was taken to determine the absolute TL using terminal restriction fragment (TRF) using Telomere Length Assay Kit (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturer’s protocol. Density measurements were performed with the Image J software (NIH, Bethesda, MD). TRF Plength was calculated using the formula TRF ¼ (ODi)/ P (ODi/Li) where ODi is the chemiluminescent signal and Li is the length of the TRF at position i. Western Blotting PBMC-isolated proteins were used for Western blotting, as described previously [23]. Primary antibodies were: mouse monoclonal p16, mouse monoclonal anti-TRF2 (Calbiochem, Temecula, CA); rabbit polyclonal IgG p21, rabbit polyclonal IgG p53, mouse monoclonal b-actin (Santa Cruz Biotechnology Inc., Dallas, Texas); mouse monoclonal anti-TRF1 (Millipore, Temecula, CA). Secondary antibody (alkaline phosphatase conjugated), purchased from Calbiochem, was diluted (1:2000 in 1X TBST) and hence detected with NBT-BCIP substrate (Calbiochem). Densitometric analysis was performed by Image J software (NIH). The fold change is expressed after normalization with the loading control (b-actin). Telomerase Activity Assay Isolated PBMC (2 105 cells) from a subset of 20 randomly chosen (age–sex matched) samples used Molecular Carcinogenesis
earlier for qPCR, in each of the three study groups, were subjected to lysis for protein extraction using the Telo TAGGG Telomerase PCR ELISA kit (Roche Applied Science), following the manufacturer’s protocol. A sample is considered to be telomerase positive if the difference in absorbance of the samples and the negative control (degraded telomerase with RNase treatment) is higher than the kit recommended 0.2. The absorbance of the supplied positive control (a known telomerase positive cell line extract) should be 1.5 A450–A690 and was 2.5 in our case. Statistical Analysis The statistical analysis was done using GraphPad InStat Software (San diego, CA). Since this is a matched case-control study pairwise t-test was performed to compare the central tendencies of continuous independent variables (like age, As-content in water, urine) between the WSL, WOSL, and unexposed groups. We used logistic regression (XLSTAT Version 2013.4.05) to determine the telomere length (RTL0.5) that corresponds to a probability of 0.5 of having arsenic specific skin lesion (Figure S1). This RTL of 0.827 denoted henceforth as RTL0.5 was used as the threshold value for predicting the risk of development of skin lesion in the study participants, as measured by odds ratio (OR) where the As-exposed individuals without skin lesions were taken as referent. RESULTS Demographic Characteristics and Arsenic Exposure of the Participants The detailed demographic characteristics of the study population are summarized in Table 1. The Ascontent in the drinking water and urine were significantly higher (P < 0.001) when compared to unexposed individuals. Arsenic Exposure Induces Senescence Via the p53-p21 Pathway On measuring the extent of As-induced senescence we found that the percentage of senescent cells (Figure 1a–c) was significantly higher (P < 0.001) in the As-exposed population (8.5 1.04%) than the unexposed (2.3 0.59%). Quantitative analysis of the senescence-inducer proteins revealed that p16 was about two-fold higher in the unexposed controls than the exposed group whereas p21 and p53 was about 2.5 and 1.46-fold, respectively, higher in the exposed group than the unexposed controls (Figure 1d and e). Differential Expression of SASP Markers With the Presence of Arsenic-Induced Skin Lesions The expression of SASP markers showed that cytokines IL-6 (Figure 2a) and IL-8 (Figure 2b) was significantly higher (P < 0.001) in the WOSL group when compared to the WSL and unexposed group.
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Table 1. Demographic Characteristic of the Study Participants
Parameters Total subjects (n) Age (in years; mean SD) Gender [n (%)] Male Female Tobacco usage [n (%)] Smoker Nonsmoker Occupation [n (%)] Farmer Business Teacher Service Student Unemployed Housewife Arsenic content in mg/L [mean SD] Drinking water Urine
Arsenic-unexposed group N (%)
Arsenic-exposed without skin lesion group N (%)
Arsenic-exposed with skin lesion group N (%)
60 44.63 11.41
60 44.85 11.55
60 44.74 11.21
32 (53.33) 28 (46.67)
35 (58.33) 25 (41.67)
37 (61.67) 23 (38.33)
14 (23.33) 46 (76.67)
10 (16.67) 50 (83.33)
11 (18.33) 49 (81.67)
32 (53.33) 2 (3.33) 1 (1.67) 3 (1.67) 5 (8.33) 2 (3.33) 15 (25)
35 (58.33) 1 (1.67) 0 2 (3.33) 4 (6.67) 3 (5.0) 14 (23.33)
34 (56.67) 1 (1.67) 2 (3.33) 1 (1.67) 3 (5.0) 3 (5.0) 16 (26.67)
9.20 0.31 30.5 0.51
143 59.0a 318 95.7a
153 65.5a 290 83.4a
Paired t-test, Pa < 0.001 (two tailed); when compared to unexposed controls.
MMP-1 (Figure 2c) and MMP-3 (Figure 2d) levels were found to be significantly higher (P < 0.001) in the WSL group when compared to the WOSL and unexposed controls. MMP-1 and MMP-3 were also
significantly higher (P < 0.01 and P < 0.001, respectively) in the WOSL group when compared to the unexposed. The concentrations of the SASP markers are listed in .
Figure 1. Comparison of arsenic-induced senescence in the study population. (a) Arsenic exposed population shows higher presence of SA b-gal positive (blue cells) in comparison to (b) Unexposed population. (c) Graph showing the percentage of SA b-gal positive cells upon arsenic-exposure in comparison to the unexposed population. (d) Protein expression for p16, p21, p53, and b-actin from PBMC of unexposed controls, WOSL and WSL. (e) Quantitative analysis of senescence inducer proteins p16, p21 and p53 from PBMC. P < 0.01; P < 0.001[Paired t-test (Wilcoxon matched pairs test) for SA b-gal and One way ANOVA for Western blot].
Molecular Carcinogenesis
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Figure 2. Measurement of SASP factors. Expression of (a) serum IL-6 and (b) IL 8 and extracellular proteases (c) MMP-1, and (d) MMP-3. P < 0.01; P < 0.001 (One way ANOVA).
Telomere Length Alteration Is Associated With ArsenicInduced Skin Lesion Telomere length status exhibited a negative correlation with age for all the study groups (Figure 3a). The distribution of TL with age in the three study groups is shown in Figure 3b. A significant increase (P < 0.001) in TL was observed in the WSL individuals (0.899 0.084) when compared to unexposed controls (0.749 0.044) as well as WOSL individuals (0.767 0.0728). Similar results were observed when absolute TL was compared between the study groups (Table S2). However, no significant increase in TL was found in the WOSL individuals with that of the unexposed controls (Figure 3c). WSL individuals had significantly higher TL for all the age groups (Figure 3d). RTL of the WSL group was positively associated with the As-content in drinking water and urine having an R2 value of 0.890 and 0.954, respectively (Figure 3e). We found that RTL value greater than RTL0.5 was significantly associated (P < 0.0001) with an increased risk (OR: 13.75; 95% CI: 5.66–33.41) towards development of As-induced skin lesion. To know the mechanism behind the differential TL, telomerase activity was measured in the three study groups. All the study groups, unexposed (0.119 0.04), Molecular Carcinogenesis
WOSL group (0.095 0.04) and WSL (0.121 0.05) had telomerase activity less than the kit recommended threshold of 0.2 and hence none of them were considered as telomerase positive (Figure 4a). TRF1 expression was higher (P < 0.01) in the WSL individuals by about 1.3 and 1.5-fold when compared with the unexposed as well as the WOSL group. Similarly, TRF2 expression was about 4 and 4.4-fold higher (P < 0.001) in WSL when compared with the unexposed and the WOSL group respectively (Figure 4b and c). DISCUSSION Earlier reports on As-induced senescence in vitro [26,27], prompted us to carry out a population based study on the alteration of senescence and telomeric regulation using blood based biomarkers. Earlier, studies have reported that the gene expression signatures present in the peripheral blood is consistent with most of the tissue types within the body like liver, heart, kidney, lung, etc. including dermatological lesion tissues in humans [28,29]. It is also well known that telomeres and telomerase play a major role at the aging-cancer interface [9]. To the best of our knowledge, this is the first study to look into As-induced senescence in human population and
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Figure 3. Measurement of telomere length. (a) Correlation between telomere length of genomic DNA from whole blood between unexposed controls, WOSL and WSL with age using Pearson's correlation coefficient (r). (b) Distribution of telomere length with respect to age in the study groups. (c) RTL comparison in the study groups. (d) Measurement of RTL with respect to age-subgroups in the study population. (e) Correlation of RTL with arsenic content in water and urine; R2 ¼ 0.890 (for water) and R2 ¼ 0.954 (for urine). P < 0.01; P < 0.001 (One way ANOVA).
telomere length alteration, on the basis of As-induced skin lesions. Our results show that the As-exposed population, irrespective of skin lesion status had significantly Molecular Carcinogenesis
increased senescent cells than the unexposed population. Arsenic might act as a xenobiotic factor, inducing senescence. The levels of the senescence inducer proteins like p16, p21, and p53 in the WSL
ARSENIC CAUSES TELOMERIC ELONGATION
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Figure 4. Mechanism of telomere length elongation upon arsenic exposure. (a) Telomerase activity measurement from PBMC isolated protein of Unexposed, WOSL and WSL groups (t-test, P > 0.05). (b) Protein expression for TRF1, TRF2, and b-actin from lymphocyte of unexposed controls, WOSL, WSL. (c) Densitometric analysis of the telomeric proteins TRF1 and TRF2. P < 0.01; P < 0.001 (One way ANOVA).
and WOSL group shows up-regulation of the p53 pathway involving p53 and its downstream effector p21 in the exposed group but not p16. Similar results were also reported earlier by other authors involving in vitro studies with As-treatment [26]. In order to understand whether the hyper-secretory factors of senescence are playing a causal role in Asinduced carcinogenesis we assessed the expression of SASP markers [8]. We observed that there is a significant increase in the IL-6 and IL-8 levels in the WOSL group whereas the invasion markers MMP-1 and MMP-3 are significantly higher in the WSL group. In a study by Ahmed et al., the IL-8 concentration in the human cord blood plasma showed a U-shaped association, increasing consistently in the group with urinary As-content 71–211 mg/L and even higher in the group with urinary-As greater than 211 mg/L. This is in accordance with our study where the urinary-As concentration were greater than 211 mg/L [30]. Thus we can infer that although arsenic is inducing the SASP, it is not associated with As-induced skin lesions and is not the pivotal factor contributing to the development of As-induced carcinogenesis. Our results on telomere length were consistent with the already established fact that telomeres act as a biomarker for replicative aging [31], evident from the negative correlation with age. On comparing the telomere length in our three study groups we observed a strikingly significant increase in telomere Molecular Carcinogenesis
length in the WSL group when compared to WOSL and unexposed group. Telomere length increase in the WSL individuals was consistent for all the age groups. This might be an indication that arsenic has a steady effect on telomere length, independent of age. In our study, individuals having RTL greater than RTL0.5 showed a significantly increased risk of developing skin lesions with an OR: 13.75 (95% CI, 5.66–33.41), when compared with the referent. Hence, it is noteworthy, that RTL could be used for predicting the risk of As-induced skin lesions in humans. The TL in the WSL group was positively correlated with As-content in water and urine, having better correlation with urinary-As content. UrinaryAs being the best measure of recent As-exposure supports our observation [32]. Previous studies have reported that treatment of human cells with arsenic resulted in maintenance of TL [33]. Recently, a population based study, conducted on women, revealed a positive association between TL and Asexposure [34]. Depending on our results, we may hypothesize that the increase in TL in the As-induced skin lesion group may be the key factor for predisposition of this group towards development of precancerous or cancerous skin lesions. To gain a mechanistic insight into the TL maintenance in WSL group we performed the TRAP assay from peripheral blood cells and found that TL elongation was telomerase-independent. Hence, our
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observations indicate that the ALT pathway may play a role in telomeric elongation in As-exposed skin lesion cases. Since our population is chronically exposed to arsenic for at least 10 years, it is expected that the lengthening of telomeres resulted from the long-term exposure of arsenic through drinking water. The APB associated shelterin complex proteins TRF1 and TRF2 is known to mediate telomere lengthening by ALT [18,35]. The proteins involved in ALT that is, TRF1 and TRF2 were found to be significantly higher in the WSL group when compared with the WOSL as well as unexposed controls. However, no significant difference was observed between the WOSL and the unexposed group indicating that the telomeric proteins are being up-regulated in the WSL group only. Our observations are in full agreement with the previous in vitro work [36], where As-treated human gastric cancer cells showed upregulation of TRF1 and TRF2 without telomere shortening, hence contradicting earlier reports [37]. TRF1 and TRF2 proteins have been identified as essential components of APB’s whereby depletion of these proteins directly inhibited the formation of APB’s. Since, the inhibition of APB’s is reflected in the impairment of the ALT-mediated telomere mainte-
nance, levels of TRF1 and TRF2 needs to be maintained for ALT [38]. Also recently, TRF2 has been associated with telomere maintenance [39]. Senescence is associated with APB formation and earlier studies have reported increased APB formation with p21 over-expression [40]. DNA compaction renders the recombination mediated ALT inactive. However, sodium arsenite has been shown to induce histone hyper-acetylation leading to an open chromatin state [41]. It can be hypothesized that arsenic leads to increased accessibility of DNA towards the APB associated proteins like TRF1 and TRF2, promoting the ALT mediated TL elongation. In summary, our findings indicate that As-exposure induces senescence in vivo, occurring via the p53 dependent pathway, inducing downstream p21, leading to the SASP markers being elevated upon As-exposure irrespective of the skin lesion status. However interestingly, TL elongation is found to be strictly associated with the occurrence of As-induced skin lesion (Table 2). Telomere length may be used for predicting the risk of susceptibility of an individual towards development As-induced dermatological lesions. We found that the telomerase-independent telomere elongation is associated with up-regulation
Table 2. Summary of Arsenic Exposure Associated Effects in the Study Groups Parameters studied
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Telomere length Telomerase activity
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" or #: Significant change (either increase/decrease) in the WOSL or WSL group with respect to unexposed (P < 0.05). or
: Significant change (either increase/decrease) in the WOSL with respect to WSL group (P < 0.05).
—, No significant change; Down-ward arrow, decrease; Up-ward arrow, increase. Molecular Carcinogenesis
ARSENIC CAUSES TELOMERIC ELONGATION
of telomeric proteins suggesting that recombinationmediated ALTs could be the mechanism behind Asinduced tumorigenesis. Further studies are required to understand this molecular mechanism, specifically from As-induced skin cancer tissues as well as other cancer types. In due course, this may help in molecular diagnosis to detect As-toxicity. ACKNOWLEDGMENTS This work was supported by Indian Council of Medical Research (ICMR) under Indo-US collaborative program [grant number 65/15/2008-NCD-I]; and Council of Scientific and Industrial Research (CSIR) Emeritus Project [grant number 21(0885)/12/EMR-II to A.K.G.]. We are indebted to Mr. Somnath Paul, Junior Research Fellow at CSIR-Indian Institute of Chemical Biology for his helpful discussions and immense support throughout the work. We are grateful to Mr. Jayanta Das, support staff at CSIRIndian Institute of Chemical Biology for on-field collection of the precious human samples. REFERENCES 1. Mondal D, Banerjee M, Kundu M, et al. Comparison of drinking water, raw rice and cooking of rice as arsenic exposure routes in three contrasting areas of West Bengal, India. Environ Geochem Health 2010;32:463–477. 2. Guidelines for Drinking Water Quality. Health criteria and other supporting information 2. Geneva: World Health Organisation; 1996. pp. 940–949. 3. Ghosh P, Banerjee M, De Chaudhuri S, et al. Comparison of health effects between individuals with and without skin lesions in the population exposed to arsenic through drinking water in West Bengal, India. J Expo Sci Environ Epidemiol 2007;17:215–223. 4. Paul S, Das N, Bhattacharjee P, et al. Arsenic-induced toxicity and carcinogenicity: A two-wave cross-sectional study in arsenicosis individuals in West Bengal, India. J Expo Sci Environ Epidemiol 2013;23:156–162. 5. Biswas D, Banerjee M, Sen G, et al. Mechanism of erythrocyte death in human population exposed to arsenic through drinking water. Toxicol Appl Pharmacol 2008;230:57–66. 6. Jasso-Pineda Y, Díaz-Barriga F, Calderón J, et al. DNA damage and decreased DNA repair in peripheral blood mononuclear cells in individuals exposed to arsenic and lead in a mining site. Biol Trace Elem Res 2012;146:141–149. 7. Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem 2004;266:37–56. 8. Copp e JP, Desprez PY, Krtolica A, Campisi J. The senescenceassociated secretory phenotype: The dark side of tumor suppression. Annu Rev Pathol 2010;5:99–118. 9. Campisi J, Kim SH, Lim CS, Rubio M. Cellular senescence, cancer and aging: The telomere connection. Exp Gerontol 2001;36:1619–1637. 10. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 1995;92:9363–9367. 11. Karlseder J, Smogorzewska A, de Lange T. Senescence induced by altered telomere state, not telomere loss. Science 2002;295:2446–2449. 12. Calado RT, Cooper JN, Padilla-Nash HM, et al. Short telomeres result in chromosomal instability in hematopoietic cells and precede malignant evolution in human aplastic anemia. Leukemia 2012;26:700–707.
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