Concurrent urinary organophosphate metabolites and acetylcholinesterase activity in Ecuadorian adolescents

https://doi.org/10.1016/j.envres.2021.112163Get rights and content

Highlights

  • Organophosphate (OP) pesticides inhibit acetylcholinesterase (AChE) activity.

  • •Unclear associations between OP metabolites and AChE have been described previously.

  • •We measured urinary OP metabolites in 526 adolescents living in agricultural areas.

  • •OP metabolites including PNP, TCPy and MDA were inversely associated with AChE.

  • •Associations were strongest at concentrations above the 80th percentile.

Abstract

Background

Organophosphates are insecticides that inhibit the enzymatic activity of acetylcholinesterase (AChE). Because of this, AChE is considered a physiological marker of organophosphate exposure in agricultural settings. However, limited research exists on the associations between urinary organophosphate metabolites and AChE activity in children.

Methods

This study included 526 participants from 2 exams (April and July–October 2016) of ages 12–17 years living in agricultural communities in Ecuador. AChE activity was measured at both examinations, and organophosphate metabolites, including para-nitrophenol (PNP), 3,5,6-trichloro-2-pyridinol (TCPy), and malathion dicarboxylic acid (MDA) were measured in urine collected in July–October. We used generalized estimating equation generalized linear model (GEEGLM), adjusting for hemoglobin, creatinine, and other demographic and anthropometric covariates, to estimate associations of urinary metabolite concentrations with AChE activity (July–October) and AChE% change between April and July–October.

Results

The mean (SD) of AChE and AChE% change (April vs July–October) were 3.67 U/mL (0.54) and −2.5% (15.4%), respectively. AChE activity was inversely associated with PNP concentration, whereas AChE% change was inversely associated with PNP and MDA. There was evidence of a threshold: difference was only significant above the 80th percentile of PNP concentration (AChE difference per SD increase of metabolite = −0.12 U/mL [95%CI: 0.20, −0.04]). Likewise, associations with AChE% change were significant only above the 80th percentile of TCPy (AChE % change per SD increase of metabolite = −1.38% [95%CI: 2.43%, −0.32%]) and PNP -2.47% [95%CI: 4.45%, −0.50%]). PNP concentration at ≥80th percentile was associated with elevated ORs for low AChE activity of 2.9 (95% CI: 1.5, 5.7) and for AChE inhibition of ≤ -10% of 3.7 (95% CI: 1.4, 9.8).

Conclusions

Urinary organophosphate metabolites, including PNP, TCPy and MDA, particularly at concentrations above the 80th percentile, were associated with lower AChE activity among adolescents. These findings bring attention to the value of using multiple constructs of pesticide exposure in epidemiologic studies.

Introduction

Organophosphate pesticides are widely used in agriculture and inhibit the activity of acetylcholinesterase (AChE) (Taylor, 2011), an enzyme that regulates neurotransmission in the central and peripheral nervous systems (Soreq, 2001), and is a well-established biomarker of effect for organophosphate exposure (Lionetto et al., 2013). The inhibition of AChE leads to the buildup of acetylcholine, potentially leading to the acute signs and symptoms of clinical organophosphate toxicity with effects on various organs such as the brain, heart, skeletal muscle, and lungs (Sultatos, 1994).

Controlled experimental studies in animals and in-vitro have described positive associations between organophosphate biomarkers and inhibition of circulating red blood cells and brain AChE activities (Banerjee et al., 1991; Eskenazi et al., 1999; Maxwell et al., 2006; Pereira et al., 2014). However, there is a limited body of literature from population-based studies that have characterized the associations between urinary organophosphate metabolite concentrations and AChE activity. This void is especially noticeable in non-occupational and pediatric populations. Some studies of pregnant women (Naksen et al., 2015) and children (Grandjean et al., 2006) did not find significant associations between AChE activity and dialkyl phosphates (DAPs), which are non-specific urinary organophosphate metabolites (Sudakin and Stone, 2011). However, studies of agricultural workers in Egypt reported significant inverse associations between 3,5,6-trichloro-2-pyridinol (TCPy), a specific metabolite for chlorpyrifos, and AChE activity in adults (Crane et al., 2013), whereas in adolescents there was evidence of a threshold, in which negative associations were observed but only among those with concentrations above 3161 μg of TCPy/g creatinine (Farahat et al., 2011).

Organophosphate pesticides have relatively short half-lives of about 24 h (±a few hours depending on the compound) (Bouchard et al., 2003; Griffin et al., 1999; Nolan et al., 1984), which reduce the exposure window that urinary metabolite concentrations reflect (which is approximately 3–6 days, considering that 94–97% of substances are excreted within this time period which equates to about 4–5 half-lives (Hallare and Gerriets, 2020)), and contribute to the substantial variability of such measures (greater within individual variability than between individuals (Bradman et al., 2013; Griffith et al., 2011)). Conversely, AChE activity reflects a much wider exposure window (enzymatic inhibition can last for 82 days if inhibited irreversibly as occurs by exposures with many organophosphate pesticides (Mason, 2000)) and, albeit less sensitive and specific, is a more stable marker of exposure than urinary metabolites (i.e., relatively low intraindividual variation over time (Bradman et al., 2013; Lefkowitz et al., 2007)). Besides AChE inhibition due to organophosphate pesticides, AChE activity can be inhibited by carbamate insecticides, nerve agents such as tabun, sarin, and soman, the repellent N,N-diethyl-3-methylbenzamide (DEET) and cholinesterase-inhibitor medications such as neostigmine, physostigmine, pyridostigmine or rivastigmine. (Abd-Ella et al., 2015; Corbel et al., 2009; Taylor, 2011; Trang and Khandhar, 2021). Additionally, in pediatric populations, age is positively associated with AChE activity (Suarez-Lopez et al., 2012).

Because of the disparate characteristics of these two constructs of organophosphate pesticide exposures (AChE and organophosphate urinary metabolites), and the limited and conflicting data characterizing the association between such constructs, it is important to characterize their relationship within population-based studies and using more specific urinary biomarkers of organophosphate exposure.

In this study, we investigate the associations between erythrocytic AChE activity and specific urinary metabolite concentrations for various organophosphate pesticides (cholinesterase inhibitors), including malathion, chlorpyrifos, diazinon and parathion among adolescents living in agricultural settings in the Andes of Ecuador. We hypothesized that greater concentrations of organophosphate urinary metabolites were associated with A) lower AChE activity in a subset of participants that had both AChE activity and urinary organophosphate metabolite concentrations measured in biospecimens collected on the same day; and B) greater AChE inhibition (a more negative percent change) between 2 examinations in a subset of participant who, in addition to having concomitant AChE and urinary metabolite concentrations, had an additional AChE measurement a few months prior.

Section snippets

Study description

The study of Secondary Exposures to Pesticides among Children and Adolescents (ESPINA) is a prospective cohort focused on understanding the effects of pesticide exposure on the development of children and adolescents residing in Pedro Moncayo County, Pichincha, Ecuador. Approximately one-fifth (Suarez-Lopez et al., 2012) of adults are employed in the floriculture industry in Pedro Moncayo, which is one of the main sites of flower production in Ecuador. A variety of insecticides are used during

Participant characteristics

The mean age was 14.5 years, 50.9% were female, 21.9% were indigenous (Table 1). We observed inverse associations of the percent indigenous participants with PNP urinary concentrations, whereas positive associations were observed with age and creatinine. There were no statistically significant differences in participants' sex, BMI-for-age z-score, height-for-age z-score, July/Oct AChE or hemoglobin by quartiles of PNP (Table 1: summer AChE). The subset of participants examined in both April and

Discussion

ESPINA is among the largest studies in adolescents investigating the effects of pesticide exposures on health. We obtained evidence in favor of our hypothesis considering that we observed an inverse association between all analyzed specific urinary organophosphate metabolites and AChE activity among a group of adolescents living in agricultural communities in the Ecuadorian Andes. The strongest associations were observed between PNP and both concurrent AChE activity and AChE% change between two

Conclusion

Our study provides novel toxicological data that quantifies the amount of AChE inhibition in relation to urinary organophosphate metabolite concentrations among adolescents living in agricultural settings. Greater urinary concentrations of PNP, TCPy and MDA were associated with lower AChE activity, particularly at concentrations above the 80th percentile. The strongest associations were observed between PNP and both concurrent AChE activity and AChE change between two time periods. These

Credit author statement

Ana Skomal: Conceptualization, Methodology, Writing - Original Draft. Jasen Zhang and Kun Yang: Statistical analysis, Software. Jessica Yen: Review & Editing. Jose Suarez-Torres: Review & Editing, Visualization, Project Administration, Data Collection. Dolores López-Paredes: Investigation, Supervision, Review & Editing, Data Collection. Antonia Calafat and Maria Ospina: Pesticide quantification, writing - Review & Editing. Danilo Martinez: Review & Editing, Data Collection. José R.

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention or the National Institutes of Health. Use of trade names is for identification only and does not imply endorsement by the CDC, the Public Health Service, or the US Department of Health and Human Services.

Declaration of competing interest

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.

Acknowledgments

Research reported in this publication was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Numbers (R01ES025792, R01ES030378, R21ES026084, U2CES026560). We thank ESPINA study staff, Fundación Cimas del Ecuador, the Parish Governments of Pedro Moncayo County, community members of Pedro Moncayo and the Education District of Pichincha-Cayambe-Pedro Moncayo counties for their contributions and support on this project. We also

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