Organophosphate insecticide exposure and telomere length in U.S. adults

Journal Pre-proof Organophosphate insecticide exposure and telomere length in U.S. adults

Jeongwon Ock, Junghoon Kim, Yoon-Hyeong Choi PII:

S0048-9697(19)35985-6

DOI:

https://doi.org/10.1016/j.scitotenv.2019.135990

Reference:

STOTEN 135990

To appear in:

Science of the Total Environment

Received date:

16 July 2019

Revised date:

26 November 2019

Accepted date:

6 December 2019

Please cite this article as: J. Ock, J. Kim and Y.-H. Choi, Organophosphate insecticide exposure and telomere length in U.S. adults, Science of the Total Environment (2018), https://doi.org/10.1016/j.scitotenv.2019.135990

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© 2018 Published by Elsevier.

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Organophosphate Insecticide Exposure and Telomere Length in U.S. Adults Jeongwon Ocka, Junghoon Kima,1, Yoon-Hyeong Choi a,b*

Running Title: Organophosphate Insecticides and Telomere Length Author Affiliation Department of Preventive Medicine, Gachon University College of Medicine, Incheon,

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Republic of Korea

Gachon Advanced Institute for Health Sciences and Technology, Incheon, Republic of Korea

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Present address: Department of Ocean Physical Education, Korea Maritime and Ocean

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*Corresponding author: Yoon-Hyeong Choi, PhD

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University, Busan, Republic of Korea

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Department of Preventive Medicine, Gachon University College of Medicine, 155, Gaetbeolro, Yeonsu-gu, Incheon, Republic of Korea 21999 Phone: +82-32-899-6586 Fax: +82-32-468-2154

E-mail: [email protected]

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ABSTRACT Background: Organophosphate insecticides have been widely used for more than 30 years, and are reported to be associated with various age-related chronic diseases. While shortening of telomere length has been considered as a marker of cellular aging, only a few small studies have been conducted to examine any difference of telomere length in workers exposed to organophosphates versus controls. Epidemiologic studies of the dose-response associations

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between environmental organophosphate exposure and telomere length in the general

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population are few.

Objective: This study aimed to evaluate the association between levels of organophosphate

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insecticide exposure and telomere length in the general population.

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Methods: We analyzed data for 1,724 participants aged 20 years or more from the National

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Health and Nutrition Examination Survey 1999─2002. Organophosphate insecticide exposure was estimated using measures of urinary concentrations for 3,5,6-trichloro-2-pyridinol

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(TCPY) and six non-specific dialkyl phosphate metabolites, e.g., diethyl thiophosphate

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(DETP). Multiple linear regression was conducted to assess the association between organophosphate exposure and telomere length. Results: After controlling for sociodemographic and physical factors and urinary creatinine, participants in the second quartile for urinary TCPY had 0.06 (95% CI: 0.02─0.10) T/S ratio shorter telomere length than those in the lowest quartile. By contrast, participants in the second and third tertiles of urinary DETP had 0.08 (95% CI: 0.02─0.14) and 0.06 (95% CI: 0.01─0.11) T/S ratio longer telomere length than those in the lowest tertile. For other five metabolites, there was no association with telomere length. Conclusions: Levels of environmental exposures to certain organophosphate insecticides may be linked to altered telomere length in adults in the general population. Although our findings may need to be replicated, we provide the first evidence that environmental exposure

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to organophosphates may contribute to the alteration of telomere length, which is potentially related to biological aging and to the development of various chronic diseases.

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Keywords: Organophosphate insecticide, Telomere length, NHANES, Epidemiology, Ageing

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Abbreviations BMI, body mass index; CDC, Centers for Disease Control and Prevention; CL, confidence level; CRP, C-reactive protein; CVD, cardiovascular diseases; DAP, dialkyl phosphate; DEDTP, diethyl dithiophosphate; DEP, diethyl phosphate; DETP, diethyl thiophosphate; DMDTP, dimethyl dithiophosphate; DMP, dimethyl phosphate; DMTP, dimethyl thiophosphate; EPA, Environmental Protection Agency; FDA, Food and Drug Administration;

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GM, geometric means; LOD, limits of detection; MEC, Mobile Examination Center; NCHS,

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National Center for Health Statistics; NHANES, National Health and Nutrition Examination

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Survey; PCR, polymerase chain reaction; PSUs, primary sampling units; SE, standard error; TCPY, 3,5,6-trichloro-2-pyridinol; TDS, Total Diet Study; TERC, template containing RNA

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components; TERT, telomerase reverse transcriptase

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1. INTRODUCTION Organophosphate insecticides have been widely used for more than three decades in the U.S. and account for almost 30% of global insecticide market sectors (i.e., agricultural, home and garden, industry, commercial, and government) (EPA, 2017). Exposure to organophosphate insecticides typically occurs through multiple pathway including inhalation, ingestion, or direct contact to the skin. Although the Environmental Protection Agency (EPA)

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has restricted residential use of most organophosphate insecticides, e.g., chlorpyrifos and

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diazinon, because of their toxicity as potent acetylcholinesterase inhibitors (Barr et al., 2011), the metabolites of organophosphate insecticides are still detected in urine samples from the

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U.S. general population (CDC, 2016). Organophosphate insecticides induce free radical

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production and consequent lipid peroxidation (Altuntas et al., 2002; Akhgari et al., 2003) that

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are related to DNA damage (Dizdaroglu et al., 2002; Kang, 2002). Previous studies report that exposure to organophosphate insecticides was associated with chronic diseases such as

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cancer (Freeman et al., 2005; Weichenthal et al., 2010), diabetes (Montgomery et al., 2008),

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cardiovascular disease (Abdullah et al., 2011), Parkinson’s disease (Manthripragada et al., 2010), and Alzheimer’s disease (Hayden et al., 2010). Telomeres are DNA-protein complexes located at the ends of chromosomes where they protect chromosomes against genome instability (Blackburn, 2005). Telomeres are typically shortened with biological aging, decreasing by 20─200 base pairs for each cell division due to the end-replication problem (Levy et al., 1992; Benetos et al., 2001; Houben et al., 2008). In terms of the cell environment, increased oxidative damage and inflammatory activity accelerate telomere shortening (von Zglinicki, 2002; O'Donovan et al., 2011). Yet telomeres can be lengthened under certain circumstances, and telomerase — a ribonucleoprotein enzyme composed of two components, telomerase reverse transcriptase (TERT) and a template containing RNA components (TERC) — controls the maintenance and elongation of

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telomeres (Blackburn, 2005). Telomere length, a cellular marker of aging, may predict an individual’s health condition and is reported to be associated with various aging-related chronic diseases (Mather et al., 2010; Shammas, 2011; Xi et al., 2013). Several epidemiologic studies link telomere length to various environmental contaminants that are known to influence oxidative stress and inflammation, but study results are inconsistent (Zhao et al., 2018): some studies show shortened telomere length (e.g., for traffic

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pollution, N-Nitrosamines, and lead) (Hoxha et al., 2009; Li et al., 2011; Wu et al., 2012; Lee

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et al., 2017) while others show increased telomere length (e.g., for arsenic, particulate matter, and persistent organic pollutants) (Dioni et al., 2011; Ameer et al., 2016; Mitro et al., 2016).

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We thereby hypothesized that exposure to organophosphate insecticides may affect

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changes in length of telomeres in the general population. Only a few small studies have been

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conducted to examine an association of telomere length and human exposure to organophosphates in occupational settings. Andreotti et al. (2015) reported that diazinon and

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malathion, two kinds of organophosphate insecticides, were significantly associated with

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shorter telomeres among 568 male insecticide applicators who participated in the Agricultural Health Study. A study by Zeljezic et al. (2015) investigated telomere length among 30 Croatian agricultural workers exposed to three organophosphate insecticides (fosetyl, chlorpyrifos, and dimethoate) and 30 controls, but failed to observe differences in telomere length between the two groups. Duan et al. (2017) investigated telomere length among 180 Chinese workers with long-term exposure to omethoate, a kind of organophosphate insecticides, and 115 controls, and observed longer telomere length in exposed workers. A study by Kahl et al. (2016a) among 62 Brazilian tobacco farmers with pesticide handling (pesticide mixture, not specific to organophosphate) and 62 controls observed shorter telomere length in tobacco farmers; their subsequent study supported previous findings through a biological system approach (Kahl et al., 2016b). However, these studies assessed

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organophosphate exposure using a questionnaire (i.e., ever use or not) and therefore could not examine the dose-response relationship between telomere length and concentrations of organophosphate metabolites. In the present study, we examined exposure to organophosphate insecticides, estimated by urinary concentrations for 3,5,6-trichloro-2pyridinol (TCPY), a specific metabolite of chlorpyrifos and chlorpyrifos-methyl, and for six non-specific dialkyl phosphate (DAP) metabolites, and telomere length as assessed in blood

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leukocyte, a proxy measure for various organ tissues (Houben et al., 2008), in a sample of

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adults representative of the general population using the National Health and Nutrition Examination Survey (NHANES) of 1999─2002 that provides data on telomere length for

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public use.

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2. METHODS 2.1. Study population NHANES was begun in the 1960s as an ongoing cross-sectional survey designed to evaluate the health and nutritional status of the U.S. general population (CDC, 2014). NHANES is conducted by the National Center for Health Statistics (NCHS) of the Centers for Disease Control and Prevention (CDC) using a complex, multistage, and probability-

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sampling design. The NHANES sampling procedure follows these steps: 1) the primary

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sampling units (PSUs) are selected and divided into multiple segments, 2) household samples are randomly drawn from the listed households within each segment, and 3) individuals are

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selected as NHANES participants from the selected households (Curtin et al., 2012).

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The present study used two consecutive cycles of NHANES 1999─2000 and NHANES

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2001─2002. Urinary organophosphate insecticide metabolite concentrations of 5,277 individuals were measured from among the 10,291 individuals aged 20 years or older in

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NHANES 1999─2002. DNA samples sufficient to measure telomere length were available

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for 2,104 adults who provided blood samples and consented to their use for future genetic research. Participants with missing data on urinary concentrations of organophosphate insecticide metabolites (n=90) and telomere length (n=2) were excluded from the present study. Further, those missing data on education level, smoking status, alcohol consumption, body mass index (BMI), hypertension, diabetes, cardiovascular diseases (CVD; including stroke, heart attack, or heart disease), or cancer were excluded from the present study (n=288), which resulted in 1,724 participants in the final analysis. All data was approved by the NCHS Research Ethics Review Board, and documented consent was obtained from participants.

2.2. Telomere length measurement

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Blood samples were collected from participants at the specially-designed and equipped Mobile Examination Center (MEC) subsequent to the households’ interview, and DNA samples extracted from the blood were stored at the National Center for Environmental Health laboratories of the CDC. Telomere length ratio relative to the standard reference DNA sample (T/S ratio) was measured in the laboratory of Dr. Elizabeth Blackburn at the University of California, San Francisco, using the method of quantitative polymerase chain

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reaction (PCR) (Cawthon, 2002). Each DNA sample was analyzed three times over three

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different days. Samples were assayed on duplicate wells, resulting in six data points for each sample. Control DNA values were used to normalize between-run variability. Runs with

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failure of more than four control DNA values beyond 2.5 standard deviations from the mean

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for all analysis runs were excluded from further analysis (<6% of runs). Any potential outliers

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were identified and excluded from the calculations for each sample (<2% of samples). CDC conducted a NCHS quality-control review of DNA samples before they were added to the

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NHANES 1999─2002 public use data files.

2.3. Organophosphate insecticide metabolites measurement In order to estimate exposure to organophosphate insecticide, one of the most common insecticide in word market (EPA, 2017), we evaluated a specific metabolite of chlorpyrifos and chlorpyrifos-methyl [TCPY] and six non-specific DAP metabolites of organophosphate insecticides [dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), dimethyl dithiophosphate (DMDTP), diethyl phosphate (DEP), diethyl thiophosphate (DETP), and diethyl dithiophosphate (DEDTP)]. These six DAP metabolites represent the breakdown products of most EPA-registered organophosphate insecticides (Bravo et al., 2004) although they cannot be identified as related to any specific insecticides. Spot urine samples were collected from participants during their physical examination at

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the MEC and frozen immediately at −20°C until shipment to the National Center for Environmental Health of the CDC. Urinary metabolites of organophosphate insecticides were measured by gas chromatography-tandem mass spectrometry (TraceGC, ThermoQuest, San Jose, CA) and quantified by the isotope dilution calibration technique. Stable isotopically labeled analogues of the TCPY or DAP metabolites were spiked into the 2ml urine, respectively. TCPY analytes were incubated to liberate conjugated metabolite, and

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hydrolyzed using solid phase extraction. DAP analytes were concentrated to dryness using an

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azeotropic distillation, dissolved with acetonitrile, and derivatized to their respective chloropropyl phosphate esters. Details of metabolite analysis are described elsewhere (Bravo

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et al., 2004).

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The analytical limits of detection (LOD) for TCPY and DEP were 0.4 μg/L and 0.2 μg/L,

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respectively. The LOD of the other five organophosphate metabolites were different for NHANES 1999─2000 and NHANES 2001─2002: 0.5 and 0.58 μg/L for DMP, 0.18 and 0.4

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μg/L for DMTP, 0.08 and 0.1 μg/L for DMDTP, 0.09 and 0.1 μg/L for DETP, and 0.05 and

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0.1 μg/L for DEDTP (CDC, 2016). All organophosphate metabolites were detected at nearly 50% or more, except DMDTP (40.0%) and DEDTP (30.5%). Subjects with values below the LOD were 391 (22.7%) for TCPY, 591 (34.3%) for DETP, 731 (42.4%) for DEP, 778 (45.1%) for DMTP, 866 (50.2%) for DMP, 1,034 (60%) for DMDTP, and 1,199 (69.5%) for DEDTP. Values below the LOD were assigned a value of LOD divided by the square root of two (Hornung and Reed, 1990). Although we considered all ten metabolites of organophosphate in NHANES 1999─2002, we finally did not include three other metabolites (i.e., malathion dicarboxylic acid, 2isopropyl-4-methyl-6-hydroxypyrimidine, and para-nitrophenol) because measurements of those metabolites were available only in a small subpopulation one third the size of the final population.

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2.4. Covariates We considered the following potential confounders: age, sex, race/ethnicity (Non-Hispanic White, non-Hispanic Black, Mexican-American, or others), education level (less than high school, graduated high school, or more than high school), pack-years of cumulative cigarette smoking (none, <20, or ≥20), recent alcohol consumption (none, <20, or ≥20 grams per day),

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physical activity (<150 or ≥150 minutes per week), BMI, hypertension, diabetes, CVD,

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cancer, C-reactive protein (CRP), and creatinine. BMI was measured as weight divided by the square of height (kg/m2). Because their distributions were right skewed, BMI and CRP were

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log-transformed. Hypertension, diabetes, CVD, and cancer were assessed by self-reports of

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previous diagnosis (yes or no). Creatinine concentrations were considered as a covariate to

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adjust dilutions of spot urine samples (Barr et al., 2005). Information on agricultural occupation (yes or no) at participant’s longest job, household pest control (yes or no) in home

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or private yard in past month, and information on dietary vitamin C intake (mg) and energy

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intake (kcal) estimated using 24-hour recall were also considered as potential confounders in sensitivity analysis.

2.5. Statistical analysis.

All statistical analyses were performed using the SAS survey procedure (version 9.4; SAS Institute Inc., Cary, NC). Four-year sample weight (WTMEC4YR) were used to account for the complex survey including oversampling, survey non-response, and post-stratification following NCHS analytic guidelines (Johnson et al., 2013). In order to evaluate associations between organophosphate metabolites and telomere length, we used SURVEYREG. Classification of each organophosphate metabolite was based on distribution of their urinary concentrations (see Table 2). Urinary TCPY concentrations

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were categorized as quartiles (<0.56 μg/L; 0.56─1.7 μg/L; 1.71─3.83 μg/L; 3.84─180 μg/L) and urinary DETP concentrations were categorized as tertiles (<0.1 μg/L; 0.1─1 μg/L; 1.01─205 μg/L). We developed sequential models to evaluate the influence of confounders: Model A was adjusted for age, sex, race/ethnicity, education, and urinary creatinine; Model B was additionally adjusted for pack-years of cigarette smoke, alcohol consumption, and physical activity; and Model C was additionally adjusted for BMI, hypertension, diabetes,

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cardiovascular diseases, cancer, and C-reactive protein. Urinary concentrations of the other

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five organophosphate metabolites were categorized into two groups using LOD as a cutoff point (
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was used as a cutoff point in this study. Mean change of telomere length was estimated by

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comparing the higher organophosphate concentration groups with the lowest

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organophosphate concentration group (reference). Additionally, we examined the association between the total amount of DAP metabolites and telomere length. The unit of metabolite

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concentration was converted from mass concentration (μg/L) to molar concentration (nmol/L),

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and then summed to yield total concentration of all DAP metabolites (ΣDAPs), dimethyl alkyl phosphates (ΣDMAPs), and diethyl alkyl phosphate (ΣDEAPs), as follows: ΣDAPs=DMP+DMTP+DMDTP+DEP+DETP+DEDTP ΣDMAPs=DMP+DMTP+DMDTP ΣDEAPs=DEP+DETP+DEDTP Their concentrations were categorized as tertiles. In all analyses, a value of P <0.05 was used for statistical significance.

2.6. Sensitivity analysis. We used sensitivity analyses to examine the influence of creatinine correction,

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agricultural-related job or household pest control activity, dietary intake, and missing data of covariates. First, we modeled creatinine-corrected concentrations of TCPY and DETP to account for variability in urine dilution (i.e., dividing organophosphate concentrations by creatinine concentrations (μg-metabolite/g-creatinine)) (CDC, 2013), instead of adjusting a covariate in the model. Second, in order to account for occupational exposure to organophosphates, we examined the association between exposure to organophosphate

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insecticides and telomere length after further adjusting for agricultural occupation or not. This

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analysis was conducted with 1,717 participants who had occupational data. Third, in order to account for household exposure to organophosphates, we examined analysis after further

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adjusting for household pest control in the subpopulation of 1,698 participants who had

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household pest control data. Fourth, because diet is known to be associated with

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organophosphates exposure (Hu et al., 2016) as well as telomere length (Mazidi et al., 2017; Tucker, 2018), we examined analysis after further adjusting for dietary vitamin C (to reflect

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antioxidant vitamin) or energy intake (to reflect overall dietary status). Fifth, instead of

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excluding missing data due to non-response to covariates (n=288), we developed models including their data by replacing each missing value with multiple imputations, increasing the final study sample to a total of 2,012 subjects. We created five separate datasets to estimate imputation‐ specific regression coefficients and then pooled the estimates to get average regression coefficients using Rubin’s rules (Rubin, 2004).

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3. RESULTS Table 1 shows demographic distribution of the study population and age-standardized telomere length by participant characteristics. Overall, the mean (± SE) of age was 43.15 (± 0.60) years, and the mean of age-standardized telomere length was 1.07 (± 0.02) T/S ratio. Telomere length was shorter in participants who were older, and age-standardized telomere length was significantly different by race/ethnicity and education level.

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Table 2 presents geometric means (GM) and distribution of urinary concentrations of organophosphate insecticide metabolites. Weighted GM (± SE) of organophosphate

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metabolites was 1.51 (± 0.08) μg/L for TCPY, 1.30 (± 0.14) μg/L for DMTP, 1.07 (± 0.05)

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μg/L for DMP, 0.73 (± 0.07) μg/L for DEP, 0.37 (± 0.04) μg/L for DETP, 0.26 (± 0.02) μg/L

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for DMDTP, and 0.10 (± 0.01) μg/L for DEDTP. The weighted medians of urinary TCPY,

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DETP, DEP, and DMTP, detected at 50% or more, were 1.65, 0.50, 0.72, and 0.84 μg/L, respectively; and the weighted medians of DMP, DMDTP, and DEDTP, detected at less than

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50%, were <0.05 (LOD), <0.1 (LOD), and <0.1 (LOD) μg/L, respectively. The positive

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correlations of TCPY with DEP and DETP; DMP with DMTP, DMDTP, DEP, and DETP; DMTP with DMDTP and DEP; DEP with DETP; and DETP with DEDTP were observed (All P-values <0.05, see Supplemental Material, Figure S1). Table 3 shows the mean change of telomere length by urinary TCPY quartiles and DETP tertiles after controlling for sociodemographic and physical factors and urinary creatinine. For the TCPY quartiles, there was a non-monotonic relationship with telomere length in all sequential models. In the fully adjusted model (Model C), participants in the second quartile for TCPY had 0.06 (95% CI: 0.02─0.10) T/S ratio shorter telomere length than the first quartile, but those in the third and fourth quartiles had no significant changes in telomere length compared to the first quartile. For the DETP tertiles, there was a significant dosedependent association with longer telomere length (P for trend=0.014); In the fully adjusted

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model (Model C), participants in the second and third tertiles had 0.08 (95% CI: 0.02─0.14) and 0.06 (95% CI: 0.01─0.11) T/S ratio longer telomere length than the first tertile. Also, we modeled TCPY and DETP as creatinine-corrected concentrations instead of adjusting a covariate in the model (Supplemental Material, Table S1), and results for TCPY became not significant, and results for DETP were similar to the results when adjusting creatinine as a covariate.

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Table 4 shows the mean change of telomere length by urinary DEP, DMTP, DMP, DMDTP,

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and DEDTP levels as binomial after controlling for covariates, and we could not observe any statistical difference in telomere length between two groups.

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Additionally, we examined the association between organophosphate insecticide exposure

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and telomere length after further adjusting for agricultural occupation or household pest

those in non-adjustment models.

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control (Supplemental Material, Table S2 and S3), and those results were fairly consistent to

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We also examined the association after further adjusting for dietary intake of vitamin C or

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energy (Supplemental Material, Table S4), and those results were fairly consistent to those in non-adjustment models.

When we developed the models with multiple imputation after including missing data due to non-response to covariates, all results of a sensitivity analysis were consistent with the results from our original analyses (Supplemental Material, Table S5). Table 5 shows the mean change of telomere length by ΣDAPs, ΣDMAPs, and ΣDEAPs tertiles in the fully-adjusted models. For the ΣDEAPs tertiles, there was a significant dosedependent association with longer telomere length (P for trend=0.027); Participants in the second and third tertiles had 0.06 (95% CI: 0.01─0.11) and 0.05 (95% CI: 0.01─0.09) T/S ratio longer telomere length than the first tertile. For ΣDAPs and ΣDMAPs, we observed no significant association.

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4. DISCUSSION In a representative sample of U.S. adults using NHANES 1999─2002, we examined associations between organophosphate insecticide exposure, estimated by urinary metabolite concentrations, and telomere length. We found that urinary concentrations of TCPY were associated with shorter telomere length, while urinary concentrations of DETP were associated with longer telomere length.

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TCPY is a specific metabolite of chlorpyrifos, the most commonly used organophosphate

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insecticide. In this study, when urinary TCPY was modeled in quartiles, significantly shorter telomere length was observed in participants in the second quartile, compared to those in the

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lowest quartile. Underlying mechanisms critical to telomere shortening are oxidative stress

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and inflammation, and previous experimental studies have suggested that organophosphates

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are capable of inducing oxidative stress and inflammation. First, two in vitro studies in rats suggest that exposure to chlorpyrifos causes dose- and time- dependent increase in the levels

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of reactive oxygen species and lipid peroxidation with DNA damage in peripheral blood

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lymphocytes (Ojha and Srivastava, 2014; Ojha and Gupta, 2017). Another in vivo study in rats suggests that both acute and chronic exposures to chlorpyrifos cause a dose-dependent increase in DNA damage-related oxidative stress in liver and brain (Mehta et al., 2008). Lukaszewicz-Hussain (2010) suggests that organophosphate induces formation of reactive oxygen species via metabolism of Cytochrome P450 enzymes, which catalyze oxidation under electron transport pathway and generate reactive oxygen species. Second, a study of human fetal astrocytes observed that IL-6 protein levels, a major mediator of inflammation, was increased after exposure to chlorpyrifos (Mense et al., 2006). Furthermore, a study using male mice suggests that exposure to chlorpyrifos, compared with other organophosphates, induce a higher extent of DNA alkylation (Mostafa et al., 1983), which is known to be associated with telomere shortening (Petersen et al., 1998). Taken together, it seems plausible

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that chlorpyrifos-induced oxidative stress, inflammation, and alkylation may affect telomere length with chromosome instability and telomeric damage. Our findings are consistent with results in the Agricultural Health Study suggesting that self-reported cumulative use of diazinon, a kind of organophosphates, is associated with shorter telomere length (Andreotti et al., 2015). In addition, previous studies report that shorter telomere length is associated with development of degenerative diseases, including Alzheimer’s disease (Hochstrasser et al.,

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2012), rheumatoid arthritis (Steer et al., 2007), and osteoporosis (Valdes et al., 2007)

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In the current study, there was non-monotonic relationship in telomere length with TCPY: significant decrease in the second quartile was observed but no significant changes in the

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third and the highest quartiles of TCPY. As our null findings might be caused by

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misclassification of participant characteristics in the third and fourth quartiles, we examined

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the distribution of participant characteristics across TCPY quartiles (Supplemental Material, Table S6) and observed significantly higher levels of physical activity in the fourth quartile

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than those in the second quartile. Because physical activity is highly related to telomere

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length (Cherkas et al., 2008; Ludlow et al., 2008), we may not be able to observe changes to telomere length in the fourth quartile even after adjusting for physical activity. Also, because age is highly related with shortened telomere length, and our analyses showed significantly lower age in the second and third quartiles than in the first quartile, we considered age as an important confounding factor. Nevertheless, there might still be residual confounding we cannot capture that leads to null findings in the third and fourth quartiles. Interestingly, DETP and ΣDEAPs, modeled as tertiles in our study, were observed to be associated with significantly longer telomere length. This finding may reflect a chemicalspecific mechanism or may be due to change possibly by residual confounding. One experimental study with rabbits reported that low- and high-dose exposure to diazinon, which metabolizes to DEP and DETP, significantly increased oxidative DNA liver damage and

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telomerase activity (Tsitsimpikou et al., 2013). Another study reported that TERT expression, related to telomere maintenance, was up-regulated in human K562 cells after exposure to organophosphate insecticides (Zhang et al., 2012). High telomerase activity occurred from acute inflammation in human and mouse models, contributing to maintenance of the cell replication capacity (Weng et al., 1995; Hodes et al., 2002). Although the mechanism of organophosphate-induced telomerase activation has not been clarified, these studies suggest

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the possibility that organophosphate insecticides may affect telomerase activation. Indeed,

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several epidemiological studies have observed associations between longer telomere length and environmental exposures including particular matter (Dioni et al., 2011), arsenic (Ameer

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et al., 2016), and persistent organic pollutants (Mitro et al., 2016). Longer telomere length is

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associated with a higher risk of several cancers, in that additional cell division provides the

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opportunity to accumulate somatic mutations and promote tumorigenesis (Lan et al., 2013). Further, breast cancer (Svenson et al., 2008), lung cancer (Shen et al., 2011), pancreatic

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cancer (Lynch et al., 2013), and non-Hodgkin lymphoma (Lan et al., 2009), which are

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associated with longer telomere may be linked to organophosphate insecticide exposure. The Agricultural Health Study has reported two different results of shorter telomere length and longer telomere length with self-reported use of organophosphates. Their findings raise the possibility that shorter telomere length results from cumulative exposures, but have inconsistent findings (either shorter or longer telomeres) that result from acute exposures, and also described a possibility of cell type—dependent different findings (Andreotti et al., 2015). A Chinese case-control study that observed longer telomere length in workers exposed to organophosphates suggested that the rate of increase in telomere length may be less steep in workers with long-term exposure and explained the possibility of genetic polymorphismdependent results of such exposure (Duan et al., 2017). However, studies on organophosphate insecticides and telomerase activation or telomere length are still lacking, so one should be

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cautious when interpreting these findings. Over their lifetime, people engaged in agricultural occupations can have high levels of exposure organophosphates and various other insecticides. In order to observe the association of telomere length with urinary organophosphates that reflect short-term environmental exposures, we conducted sensitivity analysis after controlling for agricultural occupation. However, associations between urinary TCPY and DETP with telomere length were not

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changed after adjustment for agricultural occupation. Also, we considered recent household

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insecticide use (i.e., gardening) as non-occupational and recreational exposures, but associations between urinary TCPY and DETP with telomere length were not changed after

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adjusting. This suggests that organophosphate insecticides, independent of other insecticides

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or herbicides, are associated with telomere length and that the general population is exposed

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to organophosphates by various pathways, regardless of occupation or recreation. In fact, the Pesticide Residue Monitoring Program by Total Diet Study (TDS), an ongoing program of

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the Food and Drug Administration (FDA), reported that organophosphate insecticide residues

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are still commonly detected in food (FDA, 2014), a source of exposure unrelated to occupation or recreation.

In our study, urinary DMP, DMTP, DMDTP, DEP, and DEDTP had null association with telomere length, yet we cannot rule out the possibility of their association. In the current study sample, low detection rates of DMP, DMTP, DMDTP, DEP, and DEDTP (between 30.5% and 57.6%) cannot reflect variation of metabolite concentrations and therefore may not provide statistical power to observe dose-response associations. Also, even while high levels of exposure may be shown to affect telomere length, such low levels of exposure may not be sufficient to affect risk to telomere length. In sensitivity analysis, we evaluated the association between organophosphate insecticides and telomere length by another method in considering urine dilution that calculates

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organophosphate metabolite concentrations divided by urinary creatinine. Compared to results when urinary creatinine was used as a covariate, there was no association with TCPY, but the association with DETP was concordant. Because urinary creatinine levels depend not only on the individual’s body mass and kidney function, but also on demographic characteristics such as age, gender, and race/ethnicity, our results can be biased. The use of urine creatinine level as an independent variable in the multiple regression may be more

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appropriate for adjusting urine dilution and demographic differences (CDC, 2013).

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Our study had several notable strengths. First, we used biomarkers of urinary organophosphate concentrations to estimate individual exposures, which could explain low-

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to high- exposure levels and to observe dose-response association with telomere length.

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Second, using seven kinds of organophosphates metabolites provided the opportunity to

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observe different associations between each specific metabolites and telomere length. Third, our results can be interpreted at the population level because we used the NHANES data, a

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large representative sample of adults in the general population. Finally, various potential

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confounders including sociodemographic and physio-medical factors were adjusted in our analysis. This study had considerable limitations, however. First, due to the cross-sectional design of NHANES data, we cannot infer the temporal causality between organophosphate insecticide exposure and telomere length. Second, although we used various organophosphate metabolites, each metabolite is derived from a mixture of one or more organophosphate insecticides so we cannot infer an association between a specific insecticide and telomere length. Third, single urine measurements may lead to misclassification of exposure measurements. Finally, because organophosphates have a short half-life (range of hours to weeks) (CDC, 2017) and are rapidly released from the human body, we cannot explain longterm exposures.

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5. CONCLUSIONS To the best of our knowledge, this is the first epidemiologic study to evaluate the association of various urinary metabolites of organophosphate insecticides with telomere length using biological materials collected from the general population. Our findings suggest that exposure to certain organophosphate insecticides may be associated with changes in leukocyte telomere length, although associations depended on the particular organophosphate

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metabolite: shorter telomere lengths were associated with TCPY and longer telomere lengths

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were associated with DETP. Although our hypotheses may need to be elucidated in other populations, our findings in general adult population add one of the first pieces of evidence

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that exposure to organophosphate insecticides can contribute to alteration of telomere length,

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which may be a potential intermediate in biological aging and in the development of various

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chronic diseases.

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Acknowledgements This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korea Ministry of Education [grant numbers 2013R1A6A3A04059556] and was supported by the Gachon University Research Fund of 2018 [grant number GCU-2018-0321]. The authors gratefully acknowledge Jaehyun

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Oh for creating the graphical abstract.

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Conflict of interest: None

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐ The authors declare the following financial interests/personal relationships which may

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be considered as potential competing interests:

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Table 1. Age-standardized telomere length (T/S ratio) by participant characteristics. Participants Telomere lengthb Characteristics P-valuec a (n (%) ) (T/S ratio (95% CI)) 1724 (100.0) 1.07 (1.04, 1.10) Total Age (years) 20─39 754 (45.0) 1.15 (1.11, 1.19) 40─59 627 (42.6) 1.05 (1.00, 1.09) 60─79 267 (10.2) 0.95 (0.91, 0.99) ≥80 76 (2.1) 0.87 (0.79, 0.94) <0.001 Sex Males 808 (49.8) 1.06 (1.03, 1.10) Females 916 (50.2) 1.08 (1.04, 1.11) 0.162 Race/ethnicity Non-Hispanic White 900 (74.2) 1.06 (1.03, 1.10) Non-Hispanic Black 318 (10.0) 1.11 (1.05, 1.17) Mexican American 366 (6.3) 1.03 (0.99, 1.07) Other Race 140 (9.5) 1.09 (1.03, 1.16) 0.022 Education level high school 833 (56.6) 1.08 (1.05, 1.12) 0.034 Cumulative cigarette pack-years None 971 (53.3) 1.07 (1.03, 1.11) <20 536 (33.7) 1.07 (1.04, 1.11) ≥20 217 (13.0) 1.06 (1.01, 1.11) 0.717 Alcohol consumption None 1289 (70.2) 1.06 (1.03, 1.10) <20 g/day 192 (13.2) 1.10 (1.04, 1.16) ≥20 g/day 243 (16.6) 1.07 (1.03, 1.12) 0.076 Physical activity Low (<150 min/week) 1127 (60.7) 1.06 (1.03, 1.09) High (≥150 min/week) 597 (39.3) 1.08 (1.04, 1.12) 0.121 Body mass index <30 kg/m2 1186 (70.7) 1.07 (1.04, 1.11) ≥30 kg/m2 538 (29.3) 1.06 (1.03, 1.10) 0.498 Hypertension No 1288 (77.7) 1.07 (1.03, 1.11) Yes 436 (22.3) 1.07 (1.03, 1.11) 0.929 Diabetes No 1604 (94.7) 1.07 (1.04, 1.10) Yes 120 (5.3) 1.08 (1.01, 1.15) 0.613 Cardiovascular diseases No 1615 (94.7) 1.07 (1.04, 1.10) Yes 109 (5.3) 1.07 (1.01, 1.13) 0.985 Cancer No 1602 (93.1) 1.07 (1.04, 1.10) Yes 122 (6.9) 1.07 (1.00, 1.13) 0.912 C-reactive protein Low (<0.21 mg/dL)d 860 (55.0) 1.08 (1.04, 1.11) High (≥0.21 mg/dL) 864 (45.0) 1.06 (1.02, 1.10) 0.096 a Weighted percentages from survey frequency. b Telomere length was adjusted for continuous age, except in the age characteristic group. c P-value based on survey t-test for binominal groups and based on Wald F-test for categorical groups. d Cutoff point was defined as median.

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Table 2. Urinary concentrations of organophosphate insecticide metabolites (μg/L) in NHANES 1999─2002 (n=1,724). Metabolite

LOD by cycle 99─00 01─02

n (%) ≥LOD GM

Percentiles 25th

50th

75th

90th

99th

Max

TCPY

0.4

0.4

1333 (77.3)

1.51

0.08

1.36─1.69

0.52

1.65

3.70

7.02

20.95

180.00

DETP

0.09

0.1

1133 (65.7)

0.37

0.04

0.29─0.47


0.50

1.23

2.27

9.77

205.78

DEP

0.2

0.2

993 (57.6)

0.73

0.07

0.61─0.88


0.72

3.00

6.79

27.93

190.00

DMTP

0.18

0.4

946 (54.9)

1.30

0.14

1.04─1.63


6.34

26.65

120.84

670.00

DMP

0.58

0.5

858 (49.8)

1.07

0.05

0.96─1.18


2.88

6.94

30.89

516.70

DMDTP

0.08

0.1

690 (40.0)

0.26

0.02

0.21─0.31



1.17

4.90

48.37

270.00

DEDTP

0.05

0.1

525 (30.5)

0.10

0.01

0.09─0.12


0.11

0.58

1.70

34.00

SE of GM 95% CL for GM

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Abbreviations: LOD, limit of detection; GM, geometric mean; SE, standard error; CL, confidence level.

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0.84


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Table 3. Mean change (95% CIs) of telomere length (T/S ratio) by urinary concentrations of TCPY and DETP. Metabolite

n

Model Aa

Model Bb

Model Cc

TCPY quartile (μg/L) Q1 (<0.56)

429

0.00 (Reference)

0.00 (Reference)

0.00 (Reference)

Q2 (0.56─1.7)

437

-0.06 (-0.10, -0.01)

-0.06 (-0.10, -0.02)

-0.06 (-0.10, -0.02)

Q3 (1.71─3.83)

427

-0.03 (-0.07, 0.01)

-0.03 (-0.07, 0.01)

-0.03 (-0.07, 0.01)

Q4 (3.84─180)

431

0.03 (-0.03, 0.09)

0.03 (-0.03, 0.08)

0.02 (-0.03, 0.08)

P-trend

0.334

0.299

0.309

DETP tertile (μg/L) 591

0.00 (Reference)

0.00 (Reference)

0.00 (Reference)

T2 (0.1─1)

557

0.08 (0.03, 0.14)

0.08 (0.03, 0.14)

0.08 (0.02, 0.14)

T3 (1.01─205.78)

576

0.06 (0.01, 0.11)

0.06 (0.01, 0.11)

0.06 (0.01, 0.11)

0.013

0.014

P-trend

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T1 (<0.1)

0.014

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a

Model A was adjusted for age, sex, race/ethnicity, education, and urinary creatinine. Model B was adjusted additionally for pack-years of cigarette smoke, alcohol consumption, and physical activity. c Model C was adjusted additionally for body mass index, hypertension, diabetes, cardiovascular diseases, cancer, and C-reactive protein.

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Table 4. Mean change (95% CIs) of telomere length (T/S ratio) by urinary DEP, DMTP, DMP, DMDTP and DEDTP concentrations. Metabolite DEP (μg/L)
n

Telomere length

731

0.00 (Reference)

993

0.03 (-0.01, 0.07)


789

0.00 (Reference)

≥LOD (0.41─670)

935

-0.01 (-0.05, 0.03)

875

0.00 (Reference)

849

0.00 (-0.03, 0.04)

≥LOD (0.2─190)

DMP (μg/L)
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DMTP (μg/L)

DMDTP (μg/L)
689

-p

DEDTP (μg/L)
0.00 (Reference)

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1035

1269

0.00 (-0.03, 0.04) 0.00 (Reference)

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≥LOD (0.1─34) 455 0.02 (-0.04, 0.07) All models were adjusted for age, sex, race/ethnicity, education, pack-years of cigarette smoke, alcohol consumption, physical activity, body mass index, hypertension, diabetes, cardiovascular diseases, cancer, Creactive protein, and urinary creatinine. a Upper LOD was used as a cutoff point when LODs were different in NHANES 1999─2000 and NHANES 2001─2002.

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Table 5. Mean change (95% CIs) of telomere length (T/S ratio) by urinary ΣDAPs, ΣDMAPs, and ΣDEAPs concentrations. Metabolite n Telomere Length ΣDAPs (nmol/L) T1 (6.0─27.9)

574

0.00 (Reference)

T2 (27.9─92.7)

575

-0.01 (-0.05, 0.03)

T3 (92.8─4737.8)

575

-0.02 (-0.06, 0.03)

P-trend

0.477

ΣDMAPs (nmol/L) 574

0.00 (Reference)

T2 (11.6─60.0)

575

-0.01 (-0.05, 0.02)

T3 (60.1─4724.9)

575

-0.01 (-0.05, 0.03)

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T1 (4.5─11.6)

P-trend 574

T2 (5.6─21.2)

575

T3 (21.3─1661.3)

575

-p

T1 (1.5─5.6)

ro

ΣDEAPs (nmol/L)

P-trend

0.537

0.00 (Reference) 0.06 (0.01, 0.11) 0.05 (0.01, 0.09) 0.027

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All models were adjusted for age, sex, race/ethnicity, education, pack-years of cigarette smoke, alcohol consumption, physical activity, body mass index, hypertension, diabetes, cardiovascular diseases, cancer, Creactive protein, and urinary creatinine. ΣDAPs=DMP+DMTP+DMDTP+DEP+DETP+DEDTP ΣDMAPs=DMP+DMTP+DMDTP ΣDEAPs=DEP+DETP+DEDTP

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Highlights

• Exposures to organophosphate insecticides may be linked to altered telomere length. • Their associations depended on the particular organophosphate metabolite. • TCPY was associated with shorter telomere length.

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• DETP was associated with longer telomere length.

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• This is the first epidemiological evidence for their associations in general adults.