Phoxim induces neurotoxicity and intestinal damage in Caenorhabditis elegans

Introduction: Phoxim (chemical name O-α-cyanophenylamino-O, O-diethyl phosphorothioate, molecular formula C₁₂H₁₅N₂O₃PS) is classified as a high-efficiency, low-toxicity organophosphorus insecticide. Its primary mechanism of action involves inhibition of cholinesterase activity in insects, which disrupts nerve conduction, ultimately leading to paralysis and death.

Methods: The effects of phoxim exposure (0.5, 1, and 2.5 µg/mL) on survival, neurological function, and intestinal integrity in Caenorhabditis elegans (C.elegans) were investigated.

Results: Phoxim at all concentrations significantly increased the mortality rate of C. elegans. Fluorescence microscopy revealed that 2.5 µg/mL phoxim reduced dopaminergic neural processes in the BZ555 transgenic strain of C. elegans from 4 to 2, and 0.5 and 1 µg/mL phoxim accelerated amyloid beta (Aβ)-induced paralysis in the CL4176 strain, with complete paralysis observed at 32 and 36 h, respectively. FD&C Blue #1 staining demonstrated intestinal damage in 46.7% and 68.3% of C. elegans exposed to phoxim at 1 and 2.5 µg/mL, respectively. Exposure to 1 µg/mL phoxim decreased enterocyte numbers and reduced autophagic vesicles in the lgg-1::GFP strain of C. elegans from 1.8 to 1.3. qPCR analysis revealed downregulation of autophagy-related genes (vps-34, atg-13, and unc-51) by 0.53-, 0.43-, and 0.36-fold of the control levels, respectively. RNAi targeting the eat-2 gene further confirmed the impact of phoxim on cell survival through the autophagy pathway.

Discussion: Our results indicate that phoxim exposure reduces dopaminergic neuron integrity, accelerates Aβ-induced paralysis, and damages intestinal cells through inhibition of autophagy in C. elegans.

1 Introduction

Phoxim (chemical name O-α-cyanophenylamino-O, O-diethyl phosphorothioate; molecular formula C₁₂H₁₅N₂O₃PS) is classified as a high-efficiency, low-toxicity organophosphorus insecticide. Its primary mechanism of action involves the inhibition of cholinesterase activity in insects, which disrupts nerve conduction, ultimately leading to paralysis and death. In addition, phoxim inhibits host cholinesterase activity, enhances gastrointestinal peristalsis, and accelerates parasite expulsion from host organisms (Li and Liu, 2019; Wang et al., 2020). The versatility of phoxim has been demonstrated by its broad insecticidal spectrum and the ease of application. It effectively controls various lepidopteran larvae and other agricultural pests (Lin et al., 2011; Zhang et al., 2023; Yu and Li, 2015). In the aquaculture industry of China, phoxim is widely used as a treatment for parasitic infections, including species of Gyrodactylus, Dactylogyrus, and Trichodina, and it plays a crucial role in pond sanitization and disease management (Tang et al., 2015; Zhang et al., 2015; Gao et al., 2014).

Despite the beneficial applications in pest control, phoxim poses significant environmental challenges. When applied in agricultural fields, it can enter surface water systems via runoff, resulting in aquatic contamination. This contamination has become a serious constraint on the further development of the aquaculture industry in China and aquatic product exports (He et al., 2021). The ecological impact of phoxim extends to various aquatic species, with documented toxic effects on juvenile rainbow trout (Tang et al., 2015), goldfish (Shang and Zhang, 2015), black-tailed near-red bleaks (Xu et al., 2013), and large-bodied Paralichthys olivaceus (Guan et al., 2020). At the molecular level, phoxim acts as an inhibitor of the CYP3A enzyme activity, mRNA expression, and protein production in the liver of crucian carp.

C. elegans has emerged as a valuable model organism in toxicological research because of its numerous advantages, including a short life cycle, transparent body, fully sequenced genome, and a well-characterized nervous system comprising exactly 302 neurons (Okoro et al., 2021; Yu et al., 2021). The simple yet complete biological system of C. elegans makes it particularly suitable for high-throughput screening and mechanistic studies on environmental toxicants. Furthermore, approximately 60%–80% of human genes have homologs in C. elegans, enhancing the translational relevance of the findings from this model (Kaletta and Hengartner, 2006; Leung et al., 2008).

The utilization of C. elegans across genetics, neurology, and medical research is one of the most promising approaches in modern toxicological investigations (Hartman et al., 2021; DDai et al., 2025). The well-characterized stress response pathways of C. elegans, including oxidative stress, heat shock, and detoxification mechanisms, provide valuable insights into cellular and molecular responses to xenobiotics. The primary toxic mechanism of organophosphorus pesticides, such as phoxim, involves cholinesterase inhibition; however, there is growing evidence of additional pathways of toxicity, and these remain inadequately characterized. Recent studies have implicated oxidative stress, mitochondrial dysfunction, and endocrine disruption as potential secondary mechanisms underlying organophosphate toxicity (Costa et al., 2008; Slotkin and Seidler, 2009).

The aim of the present study was to comprehensively investigate the effects of phoxim on the model organism C. elegans, with an emphasis on three critical physiological processes that may be adversely affected: autophagy, intestinal barrier integrity, and neuronal function. Using transgenic strains and advanced imaging techniques, we sought to elucidate the molecular mechanisms underlying phoxim toxicity beyond cholinesterase inhibition. These findings contribute to a more comprehensive understanding of organophosphate pesticide toxicity and potentially inform the development of safer agricultural practices and improved risk-assessment strategies.

2 Materials and methods

2.1 Main instruments and reagents

The main laboratory instruments were as follows: a Ts2R inverted fluorescence microscope (Nikon, Japan), a TD-60 low-speed centrifuge (Sichuan Shuke Instrument Co., Ltd.), a TAdvanced PCR instrument (Biometra, Germany), an SE260 electrophoresis instrument (Bio-Rad, USA), a Gel DOC^TM XR + gel imaging system (Bio-Rad, USA), a LightCycler 96 real-time fluorescence quantitative PCR instrument (Eppendorf, Germany), an Allegra X low-temperature high-speed centrifuge (Beckman, USA), 40% phoxim (Shandong Essen Chemical Co., Ltd.), an RNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.), a cDNA synthesis kit (Beijing Tiangen Biochemical Technology Co., Ltd.), SYBR Green (Beijing Tiangen Biochemical Technology Co., Ltd.), a cDNA synthesis kit (Beijing Tigermark Ltd.), and SYBR Green (Beijing Polymeric Biotechnology Co., Ltd.); qPCR primers were synthesized by Shanghai Sangong Bioengineering Co., Ltd. The M9 buffer consisted of 5.8 g of disodium hydrogen phosphate heptahydrate, 3.0 g of disodium dihydrogen phosphate, 5.0 g of sodium chloride, and 0.25 g of magnesium sulfate heptahydrate dissolved and then fixed to 1 L. The buffer was sterilized. DMSO was obtained from Beijing Solarbio Biochemical Technology Co., Ltd.

2.2 C. elegans strains

Three of the four C. elegans lines (N2, lgg-1::GFP, BZ555, and CL4176) were provided by the Yunnan State Key Laboratory of Biological Resource Conservation and Utilization. They were actually derived from the C. elegans Genetic Center (CGC). The fourth line of C. elegans, N2, is a wild type. The mutant line lgg-1::GFP was genotyped as sqIs11 [lgg-1p::mCherry::GFP::lgg-1 + rol-6] for autophagic vesicles, the BZ555 genotype was genotyped as egIs1 [dat-1p::GFP] for dopamine neurons, and the CL4176 genotype was genotyped as dvIs27 [myo-3p::A-Beta (1–42)::let-8513′UTR + rol-6 (su1006)] X. After synchronization, L1 C. elegans were grown in a constant-temperature incubator at 15 °C to L4 and then transferred to a constant-temperature incubator at 25 °C to induce the production of Aβ to paralyze the C. elegans; the rate of paralysis was considered a measure of the neurotoxicity of phoxim.

2.3 C. elegans culture and synchronization

NGM (NaCl, peptone, CaCl₂, Mg₂SO₄, phosphate buffer, and agar) culture was based on incubation in a constant-temperature chamber at 20 °C, and the C. elegans were cultured until spawning. Synchronization was performed by feeding on E. coli OP50 in a 20 °C incubator for 48 h. After a large number of eggs appeared on the plate, the C. elegans and eggs were rinsed with M9 solution in a 14-mL collection tube and centrifuged at 3,000 rpm for 2 min. The supernatant was discarded, and 2 mL of C. elegans lysing solution was added with high-speed shaking for 1 min. When a large number of worms were broken up in the tube, M9 was added and fixed to 14 mL, and centrifuged at 3,000 rpm for 2 min, and the supernatant was discarded. This was repeated thrice until no sodium hypochlorite odor was present. Subsequently, 1 mL of M9 was transferred by pipette to a 6-cm plate to disperse the eggs, and the plate was placed in an incubator at 20 °C for incubation.

2.4 Acute toxicity test: calculation of LC₅₀

Following Koch’s quantitation method, NGM media of 1, 2.51, 6.31, 15.85, and 39.81 μg/mL of phoxim at 40% concentration were prepared as the mother liquor. After the C. elegans were synchronized, the eggs were collected and incubated to the L1 stage, and the L1 C. elegans were added to the normal NGM plates to grow to the L4 stage, after which they were transferred to the NGM plates with different concentrations of phoxim for culture. Between 20 and 25 synchronized individuals were placed into each plate, and each group was set up with three parallel samples. Incubation was carried out in an incubator at a constant temperature of 20 °C for 24 h, and the number of dead C. elegans was counted through observation under a microscope. The survival experiment consisted of a short-term acute exposure (24 h). The mortality rate of C. elegans was calculated, and the experiments were repeated thrice.

2.5 Measuring the mortality rate

After determining the appropriate dose through the pretest, NGM media of 0.5, 1, 2.5, and 5 μg/mL of phoxim at 40% concentration were prepared as the mother liquor. Phoxim was first dissolved in DMSO to prepare a stock solution and then diluted with M9 buffer to the desired concentrations. The final DMSO concentration in all treatments, including the control group, was maintained at 0.1%. The control group is referred to as the blank control throughout the manuscript. After the C. elegans were synchronized, the eggs were collected and incubated to the L1 stage, and the L1 individuals were added to the normal NGM plates to grow to the L4 stage and transferred to the NGM plates with different concentrations of phoxim for culturing, and 20–25 synchronized C. elegans were placed into each plate, and each group was set up with three parallel samples. The incubation was carried out in an incubator at a constant temperature of 20 °C for 24 h, the number of deaths of the C. elegans was observed and recorded under a microscope, the C. elegans mortality rate was calculated, and the experiments were repeated thrice.

2.6 Measurement of body length and width

After synchronization, C. elegans eggs were collected and hatched to obtain L1 larvae. A total of 25 L1-stage individuals were transferred to culture plates containing phoxim at concentrations of 0.5, 1, 2.5, and 5 μg/mL. The L1-stage individuals were also added to the standard NGM plates for culture. After incubation at 20 °C for 24 h, C. elegans were observed under a microscope in order to determine their body length and width. The control group is referred to as the Blank control throughout the manuscript was set up for all experiments. Each group included three parallel samples, and all experiments were repeated three times.

2.7 RNAi interference assay

The single clone of the eat-2 gene-interfering bacteria was placed into the LB liquid medium containing ampicillin-resistant bacteria and placed into a shaker at 180 rpm at 37 °C for overnight incubation. A 300-µL volume of bacterial solution was added to the NGM medium containing 1 mmol/L IPTG and 100 g/mL AMP; the mixture was then blow-dried on an ultraclean table and allowed to incubate overnight at 25 °C to induce RNA production. Once C. elegans digests the bacterium, the small RNAs expressed in the bacterium will enter the cells of C. elegans and interfere with a specific gene to reduce the expression level of the gene. The steps in the experiment are as follows: eggs were collected from synchronized C. elegans and incubated to the L1 stage, after which they were added to the ordinary NGM plate and grown to the L4 stage and then transferred to 0.5, 1, 2.5, and 5 μg/mL phoxim NGM medium culture at a C. elegans density of 20–25 worms in each plate. Three parallel samples were set up and incubated at 20 °C for 24 h. After incubation, the activity of C. elegans was observed under a microscope; the frequency of pharyngeal swallowing was recorded, along with the mortality rate of C. elegans. The experiment was repeated thrice.

2.8 BZ555 C. elegans’ nerve cord fluorescence

After synchronization of C. elegans, the eggs were collected to hatch to the L1 stage, L1 C. elegans were added to ordinary NGM plates or NGM plates with a concentration of 1 μg/mL phoxim and cultured in an incubator at 20 °C for 48 h to the L4 stage, and the preparations were observed under an inverted fluorescence microscope to observe the fluorescence intensity of C. elegans, which included secreting dopamine neurons and changes to the nerve cord. There were 20 individual C. elegans in each group, and the experiment was repeated thrice. Quantitative analysis of the average number of neuraxes in the same field of view was carried out. Confocal fluorescent images were acquired using an Olympus confocal fluorescence microscope. Image processing and quantitative analysis were performed using Fiji (ImageJ 2, version 1.54 p): 1. image preparation, 2. threshold adjustment, 3. parameter settings, 4. start measurement.

2.9 CL4176 C. elegans’ paralysis rate

L1-stage Alzheimer’s disease (AD) model C. elegans CL4176 was cultured at 15 °C in an incubator for 48 h to the L4 stage, after which the C. elegans were transferred to NGM plates with 0.5 and 1 μg/mL of phoxim and incubated at 25 °C to induce Aβ production. The rate of paralysis of the C. elegans was recorded every 4 h. Three parallel samples were set up in each group, and the procedure was repeated thrice.

2.10 FD&C blue #1 enteric staining

FD&C Blue #1 was prepared as a 10% solution using M9 mixed with an equal volume of OP50 and then set aside. L4-stage C. elegans were transferred to plain NGM plates containing the egg-laying inhibitor pentafluorouracil, 10 mg/mL oligofructose, and 10 mg/mL inulin; NGM plates containing 0.5, 1, and 2.5 μg/mL of phoxim; and NGM plates containing 1 μg/mL of phoxim +10 mg/mL of oligofructose or 1 μg/mL of phoxim +10 mg/mL of inulin. C. elegans were incubated at a constant temperature of 20 °C for 144 h for collection. Based on preliminary experiments, the 144-h pretreatment ensured that the experiments were conducted within an appropriate time window within which the C. elegans would display this phenotype. The C. elegans were resuspended with an appropriate amount of M9, 200 individuals were aspirated into a new 1.5-mL EP tube, and the C. elegans were resuspended by adding an appropriate amount of FD&C Blue #1 OP50 mixture with gentle shaking and then stained at 20 °C for 4–5 h. Intestinal staining was observed under an inverted fluorescence microscope, and the intestines were stained with an intact blue color, indicating that the intestines were not damaged and that intestinal function was normal. The blue dye of the food diffused around the intestines, which indicated that the intestines were leaking and that intestinal function was impaired. Three parallel samples were set up for each group, and the experiment was repeated thrice.

2.11 C. elegans autophagic vesicle

The lgg-1::GFP fluorescent strain of C. elegans was synchronized and collected after the eggs were incubated to the L1 stage; these individuals were then added to the normal NGM plates or NGM plates with a concentration of 1 μg/mL phoxim and cultured in an incubator at 20 °C for 48 h to the L4 stage. The number of autophagic microsomes in the intestinal cells was counted using a fluorescent inverted microscope, with 20 C. elegans per group, and the experiments were repeated thrice.

2.12 Real-time fluorescent quantitative PCR (qPCR)

After synchronization, the eggs were collected to hatch L1 C. elegans, which were then added to ordinary NGM plates and NGM plates with 1 μg/mL phoxim for incubation. The C. elegans were collected after 48 h. The total RNA of C. elegans was extracted following the steps of the RNA extraction kit from Bao Bioengineering (Dalian) Co., Ltd., and cDNA was synthesized by reverse transcription according to the steps of the Reverse Transcription Kit from Tiangen Biochemical Technology Co., Ltd. and subjected to real-time fluorescence PCR. gpd-1 was used as the internal reference gene, and the 2^−ΔΔCT method was used to calculate the total RNA of the 1 μg/mL phoxim-stained group compared with the blank control. The primer sequences are shown in Table 1.

Table 1

Table 1. Primer sequences for real-time fluorescence quantitative PCR.

2.13 Data analysis

Each of the above-described experiments were repeated thrice. Graphpad Prism 8.4.0 was used for graphing and data analysis, and t-test was used for two-by-two comparisons, with a statistically significant difference of p < 0.05; the more the *sign, the more significant the difference.

3 Results

3.1 The effect of different concentrations of phoxim on C. elegans survival

The median concentration of phoxim resulting in 50% mortality of C. elegans within 24 h (LC50) was 3.92 μg/mL (Figure 1D). According to existing research, phoxim can affect the behavior and condition of the C. elegans. We observed that with increasing concentrations of phoxim, the body length and width of C. elegans decreased (p < 0.01) (Figures 1B,C). The 24-h mortality rate of C. elegans was determined by poisoning with increasing concentrations of phoxim: 0.5, 1, 2.5, and 5 μg/mL. The corresponding mortality rates at 24 h were 17%, 38%, 57%, and 80%, compared with the blank control (containing 0.1% DMSO) (p < 0.001) (Figure 1E). To test factors that change the rate of poisoning, the mortality rate of a particular eat-2 RNAi bacterial strain C. elegans was used. This eat-2 RNAi bacterial strain C. elegans is characterized by reduced pharyngeal pumping, resulting in decreased frequency of swallowing in the pharynx. Consequently, mortality was reduced from 17% to 8%, 38% to 22%, 57% to 37%, and 80% to 50% (p < 0.05) (Figure 1F). This indicates that the eat-2 gene inhibited swallowing, leading to a reduction in the uptake of phoxim by the C. elegans and, therefore, lower mortality than that of the non-mutant C. elegans.

Figure 1

Figure 1. Toxicological effects of phoxim exposure in C. elegans. (A) Schematic of the experimental design, in which 4 C. elegans strains (N2, lgg-1::GFP, BZ555, and CL4176) were synchronized, cultured to the L4 stage, and exposed to different concentrations of phoxim for 24 h before phenotypic assessment. (B,C) Body length and width of the N2 strain of C. elegans decreased significantly with increasing phoxim concentrations. (D) Dose–response curve fitted with a logistic regression model, revealing an LC₅₀ of 3.92 μg/mL of N2. (E) Mortality rates increased markedly with higher phoxim levels. (F) eat-2 mutant showed increased mortality rates as phoxim treatment concentrations increased, but mortality rates were lower than the corresponding values in the EV control. Statistical significance is denoted by p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), with nonsignificant differences labeled as “ns.” Each dot represents an individual C. elegans or biological replicate; error bars indicate standard deviations.

3.2 Dose-dependent neurotoxicity of phoxim in C. elegans

As evidenced by the results of the experiments reported here, phoxim exerts a strong lethal effect on C. elegans and exerts a high degree of neurotoxicity. The BZ555 strain of C. elegans with the mutant gene dat-1:GFP was used to observe the changes in dopamine neurons. The CL4176 strain, a model of AD, was induced at 25 °C, which resulted in Aβ production, causing paralysis of the C. elegans. The effects of phoxim staining on the dopamine-secreting neurons and AD symptoms were assessed using the BZ555 and CL4176 strains, respectively. Neural cord changes in dopamine-secreting neurons were observed in the BZ555 strain treated with different concentrations of phoxim. Compared with the blank control, C. elegans treated with 0.5 and 1 μg/mL phoxim showed no significant changes in the number of nerve cords, whereas C. elegans treated with 2.5 μg/mL phoxim showed a reduction in the number of dopamine-secreting neurons, and the change was from 4 to 2 (p < 0.01) (Figures 2A,B). In addition, the effect of different concentrations of phoxim on the rate of paralysis in the AD model C. elegans (strain CL4176) was examined: paralysis occurred in all CL4176 C. elegans that had ingested phoxim at 0.5 and 1 μg/mL (100% of C. elegans paralyzed at 32 and 36 h) compared to no paralysis within 40 h in the blank control (p < 0.05), suggesting that Aβ-induced paralysis was significantly accelerated by the ingestion of phoxim (Figure 2C). Based on our results, we suggest that phoxim-poisoning damaged the dopamine-secreting neurons and accelerated Aβ-induced paralysis.

Figure 2

Figure 2. Neurotoxic effects of phoxim on C. elegans. (A) Fluorescence imaging of BZ555 transgenic C. elegans with dopaminergic neuron signals visibly decreasing as phoxim concentration increases, indicating neuronal damage. (B) Quantitative analysis showed a significant reduction in fluorescence intensity at 1.0 and 2.5 μg/mL, which is indicative of a dose-dependent neurotoxic effect. (C) Results of paralysis assays using CL4176 C. elegans; higher phoxim concentrations lead to a faster onset of paralysis, further indicating that phoxim impairs neuromuscular function in a concentration-dependent manner. Statistical significance is denoted by p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), with nonsignificant differences labeled as “ns.” Each dot represents an individual C. elegans or biological replicate; error bars indicate standard deviations.

3.3 Phoxim is associated with autophagy inhibition, which may contribute to C. elegans neurotoxicity mechanism

Lgg-1::GFP is a transgenic strain of C. elegans with the LGG-1 protein fused to green fluorescent protein (GFP), enabling direct visualization of autophagy via fluorescence microscopy. Autophagy can be quantified by counting GFP puncta, making this strain a standard tool for autophagy research. Unlike the wild-type C. elegans, which has an unmodified LGG-1 protein that cannot be visualized by fluorescence, the lgg-1::GFP variant allows research workers to directly observe and measure autophagy dynamics during development, aging, and disease processes (Chen et al., 2017). The pathogenic bacterium PA14 infested C. elegans and inhibited autophagy, leading to increased mortality from intestinal damage, as well as intestinal damage related to autophagy caused by the ingestion of phoxim. Observing the changes of autophagic vesicles detected in lgg-1::GFP C. elegans, compared with the blank control, the average number of autophagic vesicles in C. elegans intestinal cells was 1.8, and the average number of autophagic vesicles in the intestinal cells was 1.3 in phoxim-infected C. elegans at 1 μg/mL, which was a significant decrease in the number of intestinal cellular autophagic vesicles. The decrease in the number of autophagic vesicles was particularly significant in some C. elegans individuals, and cells that did not form autophagosomes were present (p < 0.05) (Figures 3A,B). In order to verify the inhibition of autophagy by phoxim, the transcript levels of autophagy-associated genes, namely, vps-34, atg-13, and unc-51, were detected in C. elegans treated with 1 μg/mL of phoxim. qPCR assay revealed that compared with the blank control, the level of expression of vsp-34, atg-13, and unc-51 was downregulated by 0.53-, 0.43-, and 0.36-fold, respectively (p < 0.05) (Figure 3C). Based on our results, the inhibition of vps-34, atg-13, and unc-51 expression by phoxim led to a reduction in the autophagy levels in intestinal cells.

Figure 3

Figure 3. Phoxim inhibits autophagy in C. elegans. (A) lgg-1::GFP C. elegans treated with phoxim showed autophagosomes in intestinal cells; white arrows indicate autophagosomes. (B) Changes in the number of autophagosomes in intestinal cells of C. elegans treated with phoxim. (C) Changes in the expression of vsp-34, atg-13, and unc-51 in C. elegans treated with 1 μg/mL phoxim. Statistical significance is denoted by p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), with nonsignificant differences labeled as “ns.” Each dot represents an individual C. elegans or biological replicate; error bars indicate standard deviations.

3.4 Intestinal damage induced by phoxim

The intestine of C. elegans is closely related to immune regulation, substance metabolism, the occurrence of many diseases, and the aging process of the organism; therefore, it is especially important to detect intestinal damage induced by phoxim on C. elegans. The effects of different concentrations of phoxim on the C. elegans intestines were detected by FD&C Blue #1 staining, and the mature C. elegans were stained in the intestine after being reared in media containing different phoxim concentrations for 144 h. The results showed that the intestines were damaged to different degrees by phoxim at different concentrations. Compared with 5% of C. elegans showing intestinal damage with the blank control, 46.7% and 68.3% of C. elegans intestines were damaged by phoxim staining at 1 and 2.5 μg/mL, respectively (p < 0.05) (Figures 4A,B).

Figure 4

Figure 4. Effect of phoxim on the intestinal damage of C. elegans. (A) Intestinal staining with FD&C Blue #1 dye in C. elegans treated with different concentrations of phoxim. White arrows indicate intestinal damage. (B) Percentage of intestinal leakage in worms treated with different concentrations of phoxim. (C) Intestinal staining with FD&C Blue #1 dye in C. elegans treated with different concentrations of phoxim and supplemented with soluble dietary fiber (fructo-oligosaccharides and inulin), where “-” represents “untreated” and “+” represents “treated.” (D) Percentage of intestinal leakage in worms treated with different concentrations of phoxim and supplemented with soluble dietary fiber (fructo-oligosaccharides and inulin). Statistical significance is denoted by p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), with nonsignificant differences labeled as “ns.” Each dot represents an individual C. elegans biological replicate; error bars indicate standard deviations.

Soluble dietary fiber supplements can modulate the intestinal flora diversity, protect intestinal integrity, and reduce inflammation. In order to test whether soluble dietary fiber has a protective effect on the intestinal tract during phoxim contamination, 10 g/L oligofructose or inulin (soluble dietary fiber) was added to the contamination medium, and the proportion of C. elegans with intestinal damage decreased to 12% and 15% with the 10 g/L oligofructose or inulin treatment, respectively, compared with 50% of C. elegans intestinal damage with 1 μg/mL (p < 0.05) (Figures 4C,D). Thus, dietary fiber had a protective effect against intestinal damage caused by phoxim. In conclusion, long-term intake of either high or low doses of phoxim resulted in intestinal leakage and damage in C. elegans, and soluble dietary fiber had a protective effect against phoxim-mediated intestinal damage.

4 Discussion

C. elegans serves as an ideal biological model that possesses unique advantages in genetic, neuroscience, and toxicological research. Since the 1990s, research workers have recognized the immense potential of this species as a research tool in environmental toxicological assessment Grandjean and Landrigan (2014) identified a comprehensive list of developmental neurotoxicants that are evident in C. elegans, including heavy metals, organic compounds, pesticides, and other substances. The toxicity of these substances manifests through behavioral and neurological impairments. Specifically, organophosphate pesticides function as neurotoxic agents capable of progressively weakening and ultimately eliminating normal biological behavioral functions, such as locomotion and feeding behaviors (Kim and Kang, 2021). Given that it is essential for survival, feeding serves as a primary behavioral assessment indicator in toxicological studies of C. elegans. The feeding organ of C. elegans is the pharynx, which is a simple neuromuscular pump that completes feeding via muscle contraction and relaxation. With the emergence of feeding indicators, research utilizing foraging and feeding behavior indicators for neurotoxic agents has gradually developed (Ishita et al., 2020; Sherman and Harel, 2024; Cao et al., 2023). In this study, we utilized a strain of C. elegans containing the eat-2 mutant, a gene encoding acetylcholine receptor subunits that regulate neuronal function, thereby weakening the swallowing capability of the affected C. elegans. The results showed that eat-2 RNAi-treated C. elegans exhibited significantly reduced mortality under phoxim exposure, indicating that oral ingestion is the primary route of phoxim absorption, which reduces phoxim intake and consequently increases the worm survival rates. However, it is crucial to note that the eat-2 gene not only affects feeding processes but also participates in neuromuscular function regulation, potentially causing interactive interference in paralysis and survival assessments (Gao et al., 2018). Specifically, the eat-2 gene regulates worm sensitivity to organophosphate pesticide neurotoxicity. Reduced eat-2 function might indirectly influence worm toxicant sensitivity by altering neural signal transmission rather than solely protecting it through reduced intake (Wang, 2019). Therefore, future studies should consider using more specific feeding control genes or developing more precise quantitative feeding measurement methods to better distinguish between reduced intake and neuroprotective effects.

Impaired growth and reproduction are common manifestations of neurotoxicity (Wang et al., 2023). Variations in body length can effectively reflect the developmental rate and physiological status of C. elegans (Stojanovski et al., 2022). Here, we report that as phoxim concentration increased, it progressively inhibited the growth of C. elegans. The body width and length ratio of the treated group became significantly shorter than those of the blank control, indicating that phoxim exhibits a certain level of neurotoxicity in C. elegans. Thus, we systematically evaluated the impact of monocrotophos on the nervous system of C. elegans using two transgenic models, BZ555 and CL4176. The BZ555 variant carries the dat-1:GFP reporter gene, which enables the labeling of dopaminergic neurons. Dopaminergic neurons are critical for maintaining normal motor capabilities and behavioral coordination, and their damage may explain the reduced motor function following exposure to monocrotophos. CL4176 is an AD C. elegans model in which temperature induction leads to Aβ production in muscle cells, causing C. elegans paralysis. Based on our experimental results, we believe that this finding is significant and suggests that exposure to environmental organophosphate pesticides is a potential risk factor for neurodegenerative diseases.

Autophagy is a highly conserved intracellular metabolic process involving the formation of double-membrane autophagosomes that engulf cytoplasmic components and transfer them to lysosomes for degradation (Chen et al., 2023; Liu et al., 2023; Mochida and Nakatogawa, 2022). In C. elegans, autophagy involves four enzyme complexes: a serine/threonine protein kinase complex (UNC-51 and ATG-13) inducing autophagy activity, a class-III phosphatidylinositol 3-kinase complex (BEC-1 and VPS-34) initiating vesicle formation, and two ubiquitin-like conjugation pathways (ATG-3, ATG-4, ATG-7, and LGG-1) promoting autophagosome expansion and completion. The intestine serves as the primary metabolic tissue in C. elegans and is a critical regulator of systemic environmental stability and lifespan (Baxi and de Carvalho, 2018). Our research found that upon phoxim exposure, significant intestinal barrier damage occurred, which was manifested by blue dye leakage into surrounding tissues. Molecular mechanism investigations revealed that phoxim exposure significantly reduced autophagy-related gene expression (vps-34, atg-13, and unc-51) to 0.53, 0.43, and 0.36 times of the control levels, respectively. Simultaneously, the autophagic vesicle count in lgg-1::GFP transgenic worms decreased from 1.8 to 1.3. These results suggest that phoxim induces intestinal cell dysfunction by suppressing autophagy pathways, thereby disrupting the intestinal barrier integrity. Notably, in studies of pathogen infection of C. elegans, inhibiting autophagy does not affect the accumulation of Pseudomonas aeruginosa in the intestine but instead induces intestinal necrosis (Zou et al., 2014). This finding echoes our observations, indicating that autophagy plays a crucial role in maintaining intestinal cell health and defending against harmful exogenous substances. Of course, the autophagy process assessment in this study was conducted only at a single concentration (1 μg/mL), without establishing a dose–effect relationship, making it difficult to comprehensively elucidate the dose-dependent effects of fenitrothion on autophagy pathways. The lack of time-series observations of dynamic changes in autophagy indicates that single time-point measurements may miss critical biological processes. No mechanism verification experiments for autophagy pathways were performed, such as using autophagy agonists/inhibitors for intervention. Thus, we were unable to confirm whether the observed changes were directly regulated by the autophagy pathway. This provided a foundation for subsequent in-depth investigations.

Based on the discovery of FNT-induced intestinal damage, we explored potential protective strategies. Soluble dietary fibers have been proven to have the ability to regulate intestinal microbiota diversity, protect intestinal integrity, and reduce intestinal inflammation, and are widely present in various natural plants, such as Jerusalem artichoke, wheat, barley, rye, onion, garlic, asparagus, and banana (Wang et al., 2020; Bornet and Brouns, 2002). Our research found that adding soluble dietary fibers (such as fructo-oligosaccharides or inulin) to fenitrothion-containing C. elegans culture medium effectively mitigated fenitrothion-induced intestinal damage. This protective effect may be achieved through multiple mechanisms. First, studies have shown that soluble dietary fibers such as short-chain fructo-oligosaccharides and inulin can significantly increase the relative abundance of Bifidobacterium and reduce the proportion of potential pathogenic bacteria in the intestine (Holscher et al., 2015). This change in microbial composition may alleviate fenitrothion toxicity through the following mechanisms: beneficial bacteria may participate in the biodegradation or transformation of fenitrothion (Trinder et al., 2016), and increased microbial diversity enhances intestinal ecosystem stability and resistance to exogenous toxins (Flint et al., 2017). Second, dietary fiber might reverse fenitrothion-induced inhibition of autophagy pathways by enhancing autophagy activity. Finally, short-chain fatty acids produced by dietary fibers may directly strengthen the intestinal barrier function. Studies have demonstrated that soluble dietary fibers, such as inulin, significantly improve intestinal barrier function disorders caused by various chemicals by increasing the production of SCFAs (Liu et al., 2015). From an integrated mechanism perspective, the protective effects of dietary fiber may be closely related to the restoration of autophagic function. Previous research has shown that inulin activates autophagy through the AMPK/mTOR signaling pathway, thereby alleviating intestinal damage caused by a high-fat diet (Li et al., 2023). In the C. elegans model, fructo-oligosaccharides were confirmed to upregulate the expression of the key autophagy gene lgg-1, increase autophagy flux, extend worm lifespan, and enhance resistance to environmental stress (Zhang et al., 2019). Considering our findings that fenitrothion significantly suppressed the expression of autophagy-related genes (vps-34, atg-13, and unc-51) and reduced the number of autophagosomes, soluble dietary fiber may exert protective effects by reversing this suppression.

The findings reported here reveal the mechanisms of the toxic effects of phoxim on C. elegans through multi-system assessment, including neurological damage (dopaminergic neuron degeneration and enhanced Aβ-related toxicity) and intestinal barrier dysfunction (through the inhibition of autophagy pathways). These findings expand our understanding of the mechanisms of organophosphate toxicity. Moving forward, we plan to (1) further clarify the dual role of the eat-2 gene in phoxim toxicity (reduced feeding and regulation of neuromuscular function); (2) explore in depth the molecular mechanisms of the protective effects of dietary fiber, especially its interaction with autophagy pathways; and (3) conduct translational research to evaluate the potential impacts of long-term, low-dose phoxim exposure on the mammalian nervous system and intestinal health, providing scientific evidence for the safe use of pesticides.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

Author contributions

MZ: Writing – review and editing, Writing – original draft. XZ: Writing – review and editing, Conceptualization. HB: Methodology, Writing – review and editing. QW: Conceptualization, Writing – review and editing. WZ: Writing – review and editing, Data curation.

Funding

The authors declare that no financial support was received for the research and/or publication of this article.

Acknowledgments

Acknowledgements

The authors would like to thank Editage (http://www.editage.cn) for English language editing.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declare that no Generative AI was used in the creation of this manuscript.

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