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Published in final edited form as: Toxicol Appl Pharmacol. 2023 Jun 14;473:116600. doi: 10.1016/j.taap.2023.116600

In vitro profiling of pesticides within the Tox21 10K compound library for bioactivity and potential toxicity

Deborah K Ngan 1, Menghang Xia 1, Anton Simeonov 1, Ruili Huang 1,*
PMCID: PMC10330904  NIHMSID: NIHMS1912433  PMID: 37321325

Abstract

Pesticides include a diverse class of toxic chemicals, often having numerous modes of actions when used in agriculture against targeted organisms to control insect infestation, halt unwanted vegetation, and prevent the spread of disease. In this study, the in vitro assay activity of pesticides within the Tox21 10K compound library were examined. The assays in which pesticides showed significantly more activities than non-pesticide chemicals revealed potential targets and mechanisms of action for pesticides. Furthermore, pesticides that showed promiscuous activity against many targets and cytotoxicity were identified, which warrant further toxicological evaluation. Several pesticides were shown to require metabolic activation, demonstrating the importance of introducing metabolic capacity to in vitro assays. Overall, the activity profiles of pesticides highlighted in this study can contribute to the knowledge gaps surrounding pesticide mechanisms and to the better understanding of the on- and off-target organismal effects of pesticides.

Keywords: Tox21, 10K library, in vitro assay, pesticides, high throughput screening, cytotoxicity

Introduction

Pesticides are chemical entities that are broadly used in agriculture to control pest infestation, halt unwanted vegetation, and prevent the spread of disease. Pesticides can be further classified as insecticides, herbicides, fungicides, rodenticides, fumigate, and insect repellents based on several factors including their target pests and chemical structure1. These chemicals intrinsically show a high degree of toxicity because pesticides are intended to kill, repel, or manage various pests and disease carriers. However, exposure to pesticides can be hazardous because the known modes of actions/molecular targets of pesticides are often shared between pests and non-target organisms, including humans2. Despite their beneficial uses in agriculture, pesticides pose new challenges to human health as the exposure to these xenobiotics can affect multiple systems in the body, leading to endocrine disruptions and neurological disturbances. Therefore, there is an urgent need to evaluate the potential health risks associated with exposure to pesticide chemicals.

One type of commonly used pesticides are organophosphorus pesticides which are known toxicants that affect the functioning of the nervous system. They are extensively used in agriculture as insecticides in the U.S. due to their high efficiency and broad spectrum of activity1. However, the exposure to organophosphorus pesticides increases the danger of their toxicity to humans and other non-target organisms because most pesticides are not highly species selective3. Consequently, the known modes of action of pesticides for targeting systems or enzymes in pests may be very similar to that in humans. The acute exposure of these chemicals to humans is an area of concern since the primary site of action of organophosphorus pesticides in humans is the central and peripheral nervous system since they inhibit acetylcholinesterase (AChE), the enzyme that is responsible for the hydrolysis of the neurotransmitter acetylcholine (ACh). Despite many studies that have described the toxic effects of pesticides, there remains a knowledge gap in the nature of interactions that may occur between pesticides and humans.

In an effort to improve toxicity testing and prediction, the Toxicology in the 21st Century (Tox21) consortium4–7 has developed in vitro methods with the aim to rapidly and efficiently evaluate the safety of a diverse set of compounds, including pesticides, and improve the understanding of toxicity pathways through the development of computational and predictive modeling. In previous efforts, Tox21 utilized a quantitative high-throughput screening (qHTS) platform to test a vast number of chemicals in multiple cell-based assays. The “Tox21 10K library”8 is a collection of approximately 10,000 chemical samples that have been screened for potential biological pathway disruptions that may lead to toxicity9, 10. A previous study analyzed the potential of using chemical structural information and Tox21 assay data to distinguish drugs and environmental chemicals from the Tox21 10K library11. The Tox21 10K library has been screened in nearly 80 cell-based assays in qHTS format mostly related to nuclear receptor and stress signaling pathways, with a subset of assays targeting genotoxicity, developmental toxicity, G-protein coupled receptors, and cell-death signaling. This screening effort has generated more than 102 million data points to date that have been made publicly available12, 13.

In this study, we aimed to utilize the bioactivity profiles derived from the screening of the Tox21 10K library to identify pesticide chemicals that may lead to adverse effects as well as potential targets and pathways that may be related to pesticide activity and toxicity through the assays with the largest number of pesticide actives. We also identified pesticides that require metabolic activation by comparing their activities in assays with and without xenobiotic metabolic capability. The active pesticides identified in this study can be further investigated and re-evaluated for potential on- and off-target effects for human health risk assessment.

Materials and Methods

Compound library

The U.S. Environmental Protection Agency (EPA) Pesticides Chemical Search (EPAPCS) list contains 4,038 chemicals, with 1,425 of these compounds included within the Tox21 10K library, that have been classified by the EPA as pesticidal “active ingredients” (conventional, antimicrobial, or biopesticidal agents)14. The pesticide list was obtained from the EPA CompTox Chemical Dashboard14, with entries sourced from the Pesticide Chemical Search database15 established by the EPA’s Office of Pesticide Programs.

The Tox21 10K library consists of ~10,000 (8,969 unique) compounds such as environmental and industrial chemicals, including pesticides, drugs, food additives, and cosmetic ingredients8. Within the Tox21 10K library, a total of 1,425 compounds were considered as “pesticides” according to the EPAPCS product and use categories.

In vitro assay data

The Tox21 10K library was screened against a panel of ~80 assays with 241 readouts9, 10, 16 . All data and detailed assay descriptions with target annotations are publicly available on the National Center for Advancing Translational Sciences (NCATS), part of the National Institutes of Health (NIH), website (https://tripod.nih.gov/tox/pubdata/) and PubChem database13, 17 . Several cell lines were used for the qHTS assays including human cell lines (80.88%), murine embryo fibroblast (7.35%), Chinese hamster ovary cell line (5.88%), and other (5.88%). Most of these assays cover several targets/pathways related to nuclear receptor signaling (NR, 55.90%), stress response (SR, 11.80%), cytotoxicity (8.80%), and others related to toxicity (23.50%). In addition to the primary readouts, some assays were accompanied with a cell viability readout which serves as the counter screen used to assess the integrity of cells where a loss in signal indicates cell death or cytotoxicity. The half-maximal activity (AC50) and maximal response (efficacy) values were derived from fitting the concentration-response of each compound to a four-parameter Hill equation. Compound activity was measured by initially assigning each compound as Class 1–4 based on the type of concentration-response curve observed (1.1, 1.2, 1.3, 1.4, 2.1, 2.2, 2.3, 2.4, and 3 for activators; 4 for inactive). Curve class was then converted to curve rank, an integer between 1 and 9, such that a higher rank corresponds to a compound with higher curve quality, potency, and efficacy according to the criteria previously described18, 19 . The activity outcome of a test compound was categorized based on the average curve rank and reproducibility from the triplicate runs as one of the following: active agonist/antagonist, inconclusive agonist/antagonist, inconclusive, or inactive18, 19 .

Statistical Analysis

Assay hit rates of pesticides and non-pesticides were compared using Fisher’s exact test performed within the R package version 4.1.2. Fisher’s exact test is a statistical significance test used to examine the relationship between two nominal variables20. This test was used calculate the p-value such that p < 0.05 was considered as statistically significant.

Results

Assays Significantly Enriched with Pesticide Activity

A total of 1,425 pesticides and 7,482 non-pesticide chemicals in the Tox21 10K library were previously screened against a panel of ~80 assays with 241 readouts, the data from which were included for analysis in this study. We compared the hit rates of pesticides and non-pesticides in these assays and identified the assays in which the pesticides had a significantly higher hit rate than non-pesticides (Fisher’s exact test; p < 0.05 was considered as statistically significant) (Table 1).

Table 1:

Top 20 Tox21 assays and corresponding assay targets significantly enriched with pesticide actives (in order of significance based on p-values). Hit rates were calculated for the subset of 1,425 pesticides within the Tox21 10K compound library.

# Assay Name Assay Target Hit Rate (Pesticides) P-Value
1 tox21-p450–2c9-p1 CYP2C9 47.25% 3.87 × 10−50
2 tox21-p450-2c19-p1 CYP2C19 47.40% 1.42 × 10−41
3 tox21-p450-1a2-p1 CYP1A2 42.98% 4.64 × 10−29
4 tox21-ms-ache-p2 Acetylcholinesterase (AChE) (with human microsomes) 4.85% 3.17 × 10−28
5 tox21-ms-p53-p1 P53 (with rat microsomes) 3.84% 5.12 × 10−24
6 tox21-pr-bla-antagonist-p1 Progesterone receptor (PR) 16.28% 1.92 × 10−23
7 tox21-car-agonist-p1 CAR1 (full-length receptor) 15.64% 5.80 × 10−22
8 tox21-pxr-p1 Pregnane X receptor (PXR) 26.12% 3.79 × 10−19
9 tox21-tshr-agonist-p1 Thyroid stimulating hormone receptor (TSHR) 7.53% 2.52 × 10−18
10 tox21-ms-p53-p2 P53 (with human microsomes) 2.53% 1.29 × 10−16
11 tox21-ar-mda-kb2-luc-antagonist-p2 Androgen receptor (AR) 14.84% 8.57 × 10−16
12 tox21-tshr-antagonist-p1 Thyroid stimulating hormone receptor (TSHR) 5.29% 3.90 × 10−15
13 tox21-ache-p5 Acetylcholinesterase (AChE) (biochemical) 6.51% 3.34 × 10−14
14 tox21-err-p1 Estrogen-related receptor (ERR) 10.21% 5.33 × 10−13
15 tox21-ar-mda-kb2-luc-agonist-p1 Androgen receptor (AR) 0.88% 6.75 × 10−13
16 tox21-ache-p3 Acetylcholinesterase (AChE) (cell-based) 8.47% 8.53 × 10−13
17 tox21-ks-are-p1 Nuclear factor E2-related factor 2 antioxidant response elements (Nrf2-ARE) 12.88% 1.23 × 10−12
18 tox21-trhr-hek293-p1 Thyrotropin releasing hormone receptor (TRHR) 1.66% 2.22 × 10−12
19 tox21-shh-3t3-gli3-antagonist-p1 Sonic hedgehog (SHH)/Gli1 14.48% 6.07 × 10−12
20 tox21-pr-bla-agonist-p1 Progesterone receptor (PR) 0.07% 2.47 × 10−10
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The top ten assays with significantly higher pesticides hit rates (in descending order by p-value) are: tox21-p450-2c9-p1 (p-value 3.87 × 10−50, hit rate 47.25%), tox21-p450-2c19-p1 (p-value 1.42 × 10−41, hit rate 47.40%), tox21-p450-1a2-p1 (p-value 4.64 × 10−29, hit rate 42.98%), tox21ms-ache-p2 (p-value 3.17 × 10−28, hit rate 4.85%), tox21-ms-p53-p1 (p-value 5.12 × 10−24, hit rate 3.84%), tox21-pr-bla-antagonist-p1 (p-value 1.92 × 10−23, hit rate 16.28%), tox21-car-agonist-p1 (p-value 5.80 × 10−22, hit rate 15.64%), tox21-pxr-p1 (p-value 3.79 × 10−19, hit rate 26.12%), tox21-tshr-agonist-p1 (p-value 2.52 × 10−18, hit rate 7.53%), and tox21-ms-p53-p2 (p-value 1.29 × 10−16, hit rate 2.53%). Cytochrome P450s (CYPs) are essential enzymes that play a key role in the metabolism of clinically relevant drugs21. Major CYP isoforms include CYP2C19, CYP2C9, CYP1A2, CYP2D6, and CYP3A4. A majority of the top active assays belong to the CYPs superfamily of enzymes, which aligns with the vital role of these drug-metabolizing enzymes. Furthermore, assays with metabolic capacity (tox21-ms-ache-p2, tox21-ms-p53-p1, and tox21-ms-p53-p2) ranked among the top 10 most significant assays, indicating that many pesticides required metabolic activation. The pesticide hit rates in the top 20 significant assays for pesticides, in comparison with hit rates for non-pesticides and the Tox21 compound library, can be found in Figure 1.

Figure 1:

Figure 1:

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Pesticide hit rates in the top 20 assays significantly enriched with pesticide actives, in comparison with hit rates from non-pesticides and the entire Tox21 10K library.

A notable assay that ranked in the top five assays most significantly enriched with pesticide activity was an assay that measured AChE activity with metabolic capacity introduced by adding human liver microsomes to the system22, 23. We compared pesticide activities in the AChE assay with (tox21-ms-ache-p2) and without human microsomes (tox21-ache-p5) and identified five compounds that required metabolic activation to inhibit AChE activity: ethyl p-nitrophenyl phenylphosphorothioate (EPN) (CAS# 2104-64-5), phosalone (CAS# 2310-17-0), fonofos (CAS# 944-22-9), isocarbophos (CAS# 24353-61-5), and terbufos (CAS# 13071-79-9). All compounds belong to the organophosphorus class of compounds which are effective pesticides that are used for pest control on many food crops by inhibiting AChE activity24. In recent years, organophosphorus pesticides have been under scrutiny due to toxicity concerns to humans. The potential of organophosphorus pesticides to cause neurotoxicity and affect the developing brain (neurodevelopmental effects) are major areas of concern. The in vivo neurotoxic effects of phosphorus-containing pesticides can be estimated using in vitro screening assays that measure AChE activity with and without metabolic capability22. Some compounds showed inhibition in both AChE assays with and without metabolic capacity, but they were more potent in the AChE assay with xenobiotic metabolic capability compared to the AChE assay without microsomes with at least a 10-fold difference in IC50 values (i.e., half-maximal inhibition concentration). We prioritized compounds that inhibited AChE in assays with microsomes with IC50 values < 2 μM. Our results revealed seven compounds and their corresponding IC50 values are: azinphos-ethyl (CAS# 2642-71-9, 0.25 μM), chlorpyrifos (CAS# 2921-88-2, 0.25 μM), parathion (CAS# 56-38-2, 0.74 μM), quinalphos (CAS# 13593-03-8, 1.00 μM), coumaphos (CAS# 56-72-4, 1.07 μM), dialifor (CAS# 10311-84-9, 1.41 μM), and phoxim (CAS# 14816-183, 1.66 μM). A full list of the metabolically activated compounds can be found in Table 2.

Table 2:

Compounds (name, CAS#, and pesticide class) that required metabolic activation and exhibited higher potency levels when metabolized. IC50 values of each compound are shown in a Tox21 AChE assay with (tox21-msache-p2) and without (tox21-ache-p5) microsomes.

# Compound Name CAS# Pesticide Class tox21-ache-p5 (IC50) (μM) tox21-ms-ache-p2 (IC50) (μM)
1 Ethyl p-nitrophenyl phenylphosphorothioate (EPN) 2104-64-5 Organophosphate inactive 2.00
2 Phosalone 2310-17-0 Organophosphate inactive 5.89
3 Fonofos 944-22-9 Organophosphate inactive 2.24
4 Isocarbophos 24353-61-5 Organophosphate inactive 2.82
5 Terbufos 13071-79-9 Organophosphate inactive 47.86
6 Azinphos-ethyl 2642-71-9 Organophosphate 35.48 0.25
7 Chlorpyrifos 2921-88-2 Organophosphate 79.43 0.25
8 Parathion 56-38-2 Organophosphate 24.55 0.74
9 Quinalphos 13593-03-8 Thiophosphate/Organophosphate 79.43 1.00
10 Coumaphos 56-72-4 Oligonucleotide 53.70 1.07
11 Dialifor 10311-84-9 Organophosphate 63.10 1.41
12 Phoxim 14816-18-3 Organophosphate 56.23 1.66
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Pesticide Activity and Toxicity

We compared the hit rate distribution of pesticides and non-pesticides within the Tox21 compound library (Figure 2). Pesticides exhibited more activity across all ~80 assays with an average hit rate of 6.84% when compared to their non-pesticides counterparts which had an average hit rate of 4.64%.

Figure 2:

Figure 2:

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The compound hit rate distribution of pesticides and non-pesticides. Pesticides had an average hit rate of 6.84% while non-pesticides had an average hit rate of 4.64%.

Furthermore, Figure 3 shows the distributions of pesticides in comparison with non-pesticides across different hit rates.

Figure 3a:

Figure 3a:

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Distribution of pesticides and non-pesticides by hit rate when screened against all assays. Pesticides tend to show more promiscuous activities than other compounds in the Tox21 10K library.

Both across all assays (Figure 3a) and in only the cell viability counter screens (Figure 3b), which measure cytotoxicity, the pesticide hit rate distributions are right shifted, that is, among the compounds that showed high hit rates, pesticides outnumbered non-pesticides. Non-pesticides only outnumbered pesticides among compounds with hit rates < 10%. These findings suggest that pesticides tend to show more non-specific bioactivities and are inherently more likely to be cytotoxic than non-pesticides. The majority of pesticides were active in a small portion of Tox21 assays. However, 4% of pesticides exhibited activity in more than nearly half of the assays compared to the 3% of non-pesticides counterparts. Likewise, 30% of pesticides and 23% of non-pesticides were found to be cytotoxic in more than nearly half of the assays. Pesticides that showed high activity level as well as elevated cytotoxicity should be prioritized for further toxicological evaluation.

Figure 3b:

Figure 3b:

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Distribution of pesticides and non-pesticides by hit rate when screened against only viability assays (as a measurement of cytotoxicity). Pesticides are more likely to be cytotoxic than other compounds in the Tox21 10K library.

The top five most promiscuous pesticides (in descending order by hit rate) are: triphenyl lead acetate (CAS# 1162-06-7; hit rate 57.50%), alpha-terthiophene (CAS# 1081-34-1; hit rate 53.75%), phenylmercuric chloride (CAS# 100-56-1; hit rate 45.00%), tributyltin benzoate (CAS# 4342-36-3; hit rate 45.00%), and chlorothalonil (CAS# 1897-45-6, hit rate 43.75%). Triphenyl lead acetate (CAS# 1162-06-7) is a raw chemical material that is used in pharmaceutical intermediates. Alpha-terthiophene (CAS# 1081-34-1) is the compound that is responsible for the insecticidal activity of Tagetes minuta. Alpha-terthiophene is a singlet oxygen sensitizer that exerts antifungal, insecticidal, and nematicidal properties25. Phenylmercuric chloride (CAS# 10056-1) is a mercury-based compound that is an active ingredient for pesticides including disinfectants, fungicides, and insecticides. However, phenylmercuric chloride is not currently registered by the EPA for use in the U.S. and is not recommended for use due to its toxicity and potential neurological effects26. Tributyltin benzoate (CAS# 4342-36-3) is a conventional compound that is used in antimicrobial pesticides. It acts by targeting the nuclear receptor coactivator 2 and retinoid X receptor alpha (RXR-alpha). Chlorothalonil (CAS# 1897-45-6) is a broad-spectrum, non-systemic pesticide that is widely used against mold in cereal crops and other agriculture. This chemical belongs to a large number of chlorinated organic chemicals that have been shown to be present in the Arctic, especially in air and seawater, indicating its potential for long-range transport3. The use of chlorothalonil was banned by the European Union (EU) in 2020 due to potential health risks and environmental impact, though it remains unrestricted in the U.S.27 Chlorothalonil has not been extensively studied in recent decades, however, a prior study found chlorothalonil to be highly toxic to fish by inhibiting glutathione thereby interfering with glucose oxidation28, 29.

The most cytotoxic compound was bithionol (CAS# 97-18-7) with a viability assay hit rate of 98.78%. The second most cytotoxic compound has a viability hit rate of 97.56% and was tied among several compounds including hexachlorophene (CAS# 70-30-4), mercuric chloride (CAS# 7487-94-7), mercuric iodide (CAS# 7774-29-0), mercury (II) acetate (CAS# 1600-27-7), chlorhexidine (CAS# 55-56-1), menadione (CAS# 58-27-5), dichlone (CAS# 117-80-6), methylbenzethonium chloride (CAS# 25155-18-4), triphenyl lead acetate (CAS# 1162-06-7), and triclosan (CAS# 3380-34-5). Bithionol is an organosulfur compound that is used for the treatment of tapeworm and rumen fluke infections in animals. It is an antiparasitic drug that has been shown to inhibit the growth of ovarian cancer by means of cell cycle regulation, reactive oxygen species (ROS) generation, NF-kB inhibition, and autotoxin (ATX) inhibition30. However, bithionol was withdrawn from the market for use in humans because it was found to be unsafe as it is also a potent photosensitizer with the potential to cause serious skin disorders31. Hexachlorophene, chlorhexidine, methylbenzethonium chloride, and triclosan are broadspectrum antiseptic agents used as cleansers to prevent the spread of infections. Despite these uses, both hexachlorophene and chlorhexidine were banned by the FDA due to a lack of or insufficient information to establish safety and effectiveness, with prior studies specifically showing that toxic amounts of hexachlorophene can be absorbed through the skin of humans31, 32. Mercuric chloride, mercuric iodide, and mercury (II) acetate are mercury-containing chemicals that are highly toxic and used in agriculture as pesticides and fungicides. Mercury, even in low concentrations, is known to be hazardous to human health. A ban on most pesticides with mercury has been implemented in the U.S. since the 1960s; however, high levels of contamination remain in soil and surrounding areas of prior use33. In agriculture, menadione is used to increase the growth of many plants including tomatoes and alfalfa callus. Dichlone is a fungicide originally intended for use specifically on apple trees34. Triphenyl lead acetate is a banned pesticide in the EU for plant protection and biocide.

Discussion

Pesticides include a diverse class of toxic chemicals, often having numerous modes of actions when used against targeted organisms. Despite their advantages for uses in agriculture against certain pests, the various mechanisms of toxicity of many pesticides to non-target organisms such as humans generally remain understudied. In this study, we summarized the in vitro assay data obtained from screening the Tox21 10K library against a panel of ~80 in vitro assays. By comparing the hit rates of pesticides and non-pesticides within each assay, we identified assays that were significantly enriched with pesticide actives, which may provide clues for pesticide targets or modes of action. Furthermore, we evaluated pesticide activity and cytotoxicity profiles across the Tox21 assays. The most promiscuously active as well as the most cytotoxic pesticides were also identified. A better understanding of the modes of action of pesticides may serve as an integral tool to improve the safety and efficiency of pesticides while also exploring potential new targets that can be further investigated.

A number of pesticides kill insects by targeting the nervous system, while others act as growth regulators or endotoxins. It is well-known that organochloride compounds cause neurotoxicity due to the irreversible inhibition of AChE, an enzyme located at the neuromuscular junctions and brain cholinergic synapses that is involved in the hydrolysis of ACh35. Three (tox21-ms-ache-p2, tox21-ache-p5, and tox21-ache-p3) out of the top 20 assays target AChE and were significantly enriched with pesticide activity in our study. Additionally, a number of organochloride compounds belong to the class of chemicals called endocrine-disrupting chemicals (EDCs), which interfere with endogenous hormones by binding to and activating various hormone receptors (e.g. androgen receptor (AR), progesterone receptor (PR), and estrogen-related receptor (ERR))36. Several of our assays (tox21-pr-bla-antagonist-p1, tox21-ar-mda-kb2-luc-antagonist-p2, tox21-err-p1, tox21-ar-mda-kb2-luc-agonist-p1, and tox21-pr-blaagonist-p1) that were significantly enriched with pesticide activity targeting hormone receptors, including AR, PR, and ERR. Along the same lines, pesticides have been shown to interfere with thyroid homeostasis, with different pesticide class resulting in thyroid disruption at various levels37. Despite the wide use of pesticides, in vitro evidence of thyroid disruption remains a major gap to be filled for some pesticide classes. Thyroid stimulating hormone (TSH) is produced by the anterior pituitary and is currently considered the most sensitive indicator of thyroid status37. Three significant pesticide active assays (tox21-tshr-agonist-p1, tox21-tshrantagonist-p, and tox21-trhr-hek293-p1) may aid in the detection of thyroid hormone disruption caused by pesticides.

Numerous pesticides undergo biotransformation, a detoxification process to facilitate the elimination of pesticides, by cytochrome P450 (CYP) enzymes. Since CYP genes are responsible for transcribing enzymes essential in xenobiotic metabolism, pesticides have been reported to induce CYP expression thus altering the balance of detoxification and activation38. The top three most significant assays for pesticide activity (tox21-p450-2c9-p1, tox21-p450-2c19-p1, and tox21-p450-1a2-p1) all target various CYP isoforms, reiterating that many pesticides are potent inducers for a wide range of CYPs. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are major regulators of CYP gene expression, and two of our assays (tox21-pxr-p1 and tox21-car-agonist-p1) that target for these xenobiotic-sensing nuclear receptors ranked in the top significant assays for pesticide activity, respectively.

Furthermore, these chemicals can alter the normal functioning of the endocrine system, interfering with the synthesis, transport, metabolism, and elimination of hormones. Pesticides also block the effects of several sex hormones, which may also lead to abnormal sexual development and disruption to other vital reproductive and developmental processes35. Our results also revealed the tox21-shh-3t3-gli3-antagonist-p1 assay as a significant target of pesticide activity. During embryonic development, the sonic hedgehog (SHH) protein functions as a chemical signal for cell proliferation as well as cell specialization and patterning39. While pesticides are believed to downregulate SHH signaling, there remains a need to better understand the effects of pesticides on this signaling pathway.

Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of the antioxidant response. Under normal physiological conditions, the Nrf2 proteins are kept at low levels; however, a cell may activate the Nrf2 anti-oxidant response elements (Nrf2-ARE) when triggered by oxidative stress, such as exposure to different environmental contaminants including pesticides, in an effort to regulate oxidative stress derived damages40, 41. A Tox21 assay (tox21-ks-are-p1) that targets Nrf2-ARE revealed statistical significance for pesticide activity when compared to non-pesticides, so we hope this finding may lead to a more comprehensive understanding of the oxidative stress-related toxicological targets of pesticides. A cell may also respond to oxidative stress (e.g., DNA damage) by activating p53, a tumor suppressor gene that plays key role in activating cell division and cell death42. The p53 protein was a target revealed by two of our top 20 significant assays (tox21-ms-p53-p1 and tox21-ms-p53-p2) to be enriched of pesticide activity. A prior study found that the organophosphorus pesticides parathion and malathion inhibited the p53 gene, resulting in the malignant transformation of breast cells43. Additional research is required to better understand the mechanisms of pesticides and evaluate their safety to human health, so our findings begin to bridge the role of pesticides and p53 inactivation.

Metabolic activation is a key determinant of the toxicity potential of many pesticides, e.g., some pesticides undergo biotransformation to their corresponding oxygen analogs, or oxons, which are potent AChE inhibitors44. To explore the impact of metabolism on compound activity, we compared activities in the AChE assay with and without microsomes and identified pesticides that exhibited higher potency levels when metabolized (Table 2). Here, we discuss several pesticides that are susceptible to metabolic activation and may warrant further investigation. For example, ethyl p-nitrophenyl phenylphosphorothioate (EPN) (CAS# 2104-64-5) is a notable active ingredient in pesticides that is highly active when metabolized. EPN is an insecticide of the organophosphorus class that is used to control pests as an AChE inhibitor, causing delayed neurotoxicity. Phosalone (CAS# 2310-17-0) is another organophosphate chemical that is commonly used as an insecticide and acaricide. The EU banned phosalone from its pesticide registration in 2006, and it is no longer registered as a pesticide for use in the U.S.45 Fonofos (CAS# 944-22-9) is an organophosphorus pesticide that is used primarily for corn crops, and it is listed as highly toxic to humans and animals46. Isocarbophos (CAS# 24353-61-5) is another organophosphorus pesticide insecticide and acaricide that is highly effective to control insects in agriculture. China has begun to phase out the use of several highly toxic pesticides, including isocarbophos, by prohibiting its use on vegetables, melons and fruits, tea, fungi, and Chinese herbal medicine, although it is still used on cotton and rice47. By 2024, China plans to prohibit the sale and use of isocarbophos48. Terbufos (CAS# 13071-79-9) belongs to the chemical family of organophosphates and is commonly used as an insecticide and nematicide. This compound still remains in use in the U.S. mainly on corn crops, though it is eligible for reregistration by the EPA49. Terbufos is not used or registered as a pesticide for use in several EU nations, including Finland, Norway, and Iceland3. A previous study23 also identified many of the compounds discussed above to be more potent for AChE inhibition after incubation with microsomes, thus suggesting that some organophosphorus compounds require biotransformation to be effective AChE inhibitors.

Some compounds showed inhibition in both AChE assays with and without metabolic capacity, but they were more potent in the AChE assay with xenobiotic metabolic capability. For example, azinphos-ethyl (CAS# 2642-71-9) is an organophosphorus pesticide used to protect fruit trees and crops, such as rice, from pests. It is also neurotoxicant and AChE inhibitor that was linked to health problems. As a result, azinphos-ethyl is banned in several countries including Thailand and the Philippines50, 51. Azinphos-ethyl is classified as highly hazardous (Class Ib) technical grade active ingredients by the World Health Organization (WHO)52. Chlorpyrifos (CAS# 2921-88-2) also belongs to the organophosphorus pesticide class, and it affects the nervous system by binding to AChE, thereby inhibiting the breakdown of the neural signal carrier ACh. Accumulation of ACh may lead to overstimulation of nerves and can result in paralysis, seizures, and eventual death of the insect. Chlorpyrifos was first registered for use in the mid-1960s and is currently classified as moderately hazardous (Class II) technical grade active ingredients in pesticides by the WHO3, 52. In 2022, the EPA banned the use of chlorpyrifos on any food sold in the U.S.53 In other words, food products with chlorpyrifos residue are no longer allowed and farmers may not use pesticide products containing chlorpyrifos on crops grown for any food or feed use. Parathion (CAS# 56-38-2) is classified as extremely hazardous (Class Ia) technical grade active ingredients in pesticides by the WHO52 and Acute Toxicity Category I (most toxic) by the EPA. Parathion was banned in the U.S. due to concerns to human health, especially children, and the environment that were linked to disruptions of the nervous system through AChE inhibition. Paraoxon (CAS# 311-45-5), an active metabolite of parathion, is an organophosphate oxon that is more toxic. Quinalphos (CAS# 13593-03-8) is classified as moderately hazardous (Class II) technical grade active ingredients in pesticides by the WHO52. Quinalphos is an AChE inhibitor leading to the subsequent accumulation of toxic levels of the neurotransmitter ACh. Coumaphos (CAS# 56-72-4) belongs to the oligonucleotide chemical analogue class of phosphorothioates. It is classified as a highly hazardous (Class Ib) technical grade active ingredient in pesticides by the WHO52. As with all organophosphorus insecticides, coumaphos acts on the nervous system by inhibiting the activity of AChE enzymes. Once metabolized, coumaphos becomes activated to the more toxic oxon form54. Dialifor (CAS# 10311-84-9), also referred to as dialofos, is an active ingredient believed to be obsolete or discontinued for use as pesticides by the WHO52. It is an organophosphorus insecticide used to control pests common to apples, citrus, grapes, nut trees, potatoes, and vegetables55. Phoxim (CAS# 14816-18-3) is classified as a moderately hazardous (Class II) technical grade active ingredient in pesticides52. It belongs to the organophosphorus class of pesticides and was banned in the EU in 2007 for use on crops. When metabolized, phoxim, like parathion, is oxidatively desulfurated to yield a toxic analogue that is a more potent AChE inhibitor than the parental compound56. While some pesticides have well known targets and mechanisms of action, there is still a large number of pesticides that lack sufficient toxicity information because their underlying molecular mechanisms that lead to adverse health effects remain unclear. Our Tox21 assay panel revealed several targets that reinforce known pesticide targets as well as highlighted understudied targets that may benefit from further investigation. Furthermore, the chemicals collectively discussed exemplify pesticides that require metabolic activation and exhibit higher potency levels when metabolized.

Conclusion

In this study, we examined the in vitro assay activity profiles of pesticides within the Tox21 10K library. The assays in which pesticides showed significantly more activities than non-pesticide chemicals revealed potential targets and mechanisms of action for pesticides. Furthermore, we identified pesticides that showed promiscuous activity against many targets and cytotoxicity, which warrant further toxicological evaluation. In addition, we pointed out several pesticides that required metabolic activation as well as exhibited higher potency levels when metabolized, demonstrating the importance of introducing metabolic capacity to in vitro assays for assessing pesticide toxicity. Overall, the assay targets highlighted for pesticides here may serve as a starting point to better understand the underlying biological mechanisms that are disrupted when humans are exposed to pesticides.

Highlights:

  • In vitro assay activity profiles of pesticides within the Tox21 10K library revealed potential targets and mechanisms of action for pesticides.

  • Pesticides that showed promiscuous activity against many targets and cytotoxicity were identified.

  • Differential activities observed in assays with and without metabolic capacity identified pesticides that required metabolic activation.

Acknowledgement

This work was supported by the Intramural Research Programs of the National Toxicology Program (Interagency agreement #Y2-ES-7020-01), National Institute of Environmental Health Sciences and the National Center for Advancing Translational Sciences, National Institutes of Health. The views expressed in this article are those of the authors and do not necessarily reflect the statements, opinions, views, conclusions, or policies of the National Center for Advancing Translational Sciences, the National Institutes of Health, or the United States government. Authors would like to thank Srilatha Sakamuru, Jinghua Zhao, Shuaizhang Li, Caitlin Lynch, and Li Zhang for generating the Tox21 screening data. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

Footnotes

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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.

Credit Author Statement

D.K.N.: Methodology, Formal analysis, Writing – original draft, Writing – reviewing & editing; M.X.: Supervision, Writing – reviewing & editing; A.S.: Supervision, Writing – reviewing & editing; R.H.: Conceptualization, Supervision, Writing – reviewing & editing.

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.

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