INTRODUCTION:

Chlorpyrifos (CPF) is a chlorinated organophosphate insecticide recognized for its broad-spectrum efficacy in controlling agricultural insect pests, contributing to approximately 40% of the global organophosphate consumption. The significance of CPF is particularly pronounced in Indonesia, where average import figures from 2017 to 2021 reached 19.8 million kilograms. CPF is employed for the management of various pest types, including biting, sucking, and stinging species, such as armyworms and mites, thereby playing a crucial role in crop quality maintenance².

The use of organophosphate pesticides, including CPF, is associated with several detrimental health effects, notably the induction of oxidative stress. CPF inhibits acetylcholinesterase activity, leading to the intracellular accumulation of calcium ions and subsequent mitochondrial dysfunction. This dysfunction facilitates the production of ROS, which are deleterious by-products of cellular metabolism. The active metabolite of CPF, chlorpyrifos-oxon (CPO), further exacerbates toxicity by inhibiting acetylcholinesterase, resulting in elevated acetylcholine (ACh) levels in the synaptic cleft. This cascade of events culminates in excessive cholinergic activity and alters cellular respiration, subsequently increasing ROS levels and triggering oxidative stress. The progression of oxidative stress is linked with cellular apoptosis and tissue damage³.

The oxidative stress caused by CPF reflects an imbalance between pro-oxidants and antioxidants within biological systems. CPF exposure has been shown to diminish the levels of critical antioxidants, including the enzyme superoxide dismutase (SOD). Empirical research involving goldfish has demonstrated that CPF exposure significantly reduced SOD enzyme levels after a 30-day treatment period⁴. Furthermore, CPF exposure has been correlated with increased production of ROS, notably the superoxide anion, which SOD converts to hydrogen peroxide (H2O2). Numerous studies have addressed the impact of CPF exposure on malondialdehyde (MDA) levels, with findings indicating a notable increase in MDA concentrations in ocular tissues following exposure to 12mg/kg body weight of chlorpyrifos after seven days⁵.

Autophagy, a term originating from Greek that means "self-eating," is a catabolic process that involves the breakdown and recycling of cellular materials inside lysosomes⁶. The discovery of lysosomes as cellular organelles, reported by Christian de Duve in the 1950s, paved the way for the conceptualization of autophagy. De Duve coined the term in 1963 to describe the phenomenon whereby cells merge vesicles containing proteins with lysosomes, facilitating the breakdown of these cellular proteins. The primary role of autophagy is to function as a defensive mechanism under cellular stress⁷. Autophagy encompasses a series of tightly regulated processes, including the formation of autophagosomes and autophagolysosomes and subsequent degradation. The Key regulatory mechanisms include the mechanistic target of the rapamycin (mTOR) route and the AMP-activated protein kinase (AMPK) pathway, which are crucial in monitoring cellular nutritional status⁸. While autophagy is primarily regulated at the post-translational stage, various stressors can enhance the expression of genes associated with autophagy. The transcription factor EB (TFEB), typically located in the cytoplasm, translocates to the nucleus upon phosphorylation, acting as a key regulator of these genes. This translocation triggers the activation of transcriptional pathways that manage lysosomal and autophagy-related genes. To date, over 42 genes involved in the autophagy process have been identified, highlighting the complexity and dynamic characteristics of this cellular mechanism¹¹.

Autophagy plays a role in preserving cell viability amidst cellular modifications, as evidenced by a substantial body of research implicating it in various pathophysiological conditions, including cancer, metabolic disorders, and neurodegenerative diseases. These findings position autophagy as a promising target for therapeutic interventions across various illnesses¹². Under physiological conditions, autophagy is critical for maintaining cellular homeostasis by recycling cytoplasmic proteins and organelles. The eye lens consists of specialized fiber cells that develop from epithelial progenitors and degrade organelles throughout terminal maturation. After development, these fiber cells shed their organelles, resulting in an organelle-free zone (OFZ) establishment, which regulates lens transparency¹³. Autophagy regulates the establishment of this OFZ, as disruptions in autophagic processes within lens cells can compromise their differentiation. Chronic oxidative stress results in the accumulation of ROS, which in turn can trigger apoptosis within lens cells¹⁴. Notably, ROS levels significantly escalate within 24 hours of exposure to hydrogen peroxide (H2O2), accompanied by increases in lipid peroxidation markers, such as malondialdehyde (MDA), and reductions in superoxide dismutase (SOD) enzyme activity and reduced glutathione (GSH) levels. There is a homeostatic defense mechanism during oxidative stress¹⁵. This excessive ROS production disrupts cellular homeostasis and further induces autophagic responses The mechanistic target of the rapamycin (mTOR) complex functions as a principal modulator of the autophagy pathway, exerting inhibitory effects on the serine/threonine kinase ULK1 when nutrient availability and growth factors are adequate. Conversely, oxidative stress impedes mTORC1 activity and enhances AMPK phosphorylation, resulting in mTORC1 inhibition and subsequent induction of autophagy. Chronic oxidative stress elevates p62 expression, and the ensuing aggregation of p62 with NRF2/KEAP1 facilitates the binding of NRF2/KEAP1, prompting NRF2 translocation to the nucleus. Activation of the NRF2/KEAP1/p62 signaling cascade mitigates oxidative stress and enhances the expression of antioxidant enzymes such as SOD, further reducing superoxide radicals to H2O2 and non-reactive O2. p62 accumulation is a biomarker indicating autophagy inhibition and derangements in autophagic degradation¹⁶.

During autophagic processes, cytoplasmic materials are sequestered within double-membrane vesicles, leading to autophagosome formation, then transports these materials to lysosomes for degradation. The fusion of autophagosomes with lysosomes generates single-membrane vesicles known as autolysosomes¹⁷. In this context, LC3-I undergoes lipidation with phosphatidylethanolamine (PE), yielding LC3-II, with LC3-II levels as a quantitative indicator of autophagosome formation.

Caspase-3 emerges as a critical mediator in the apoptotic cell execution death¹⁸ and represents a potential molecular switch that facilitates crosstalk between the autophagic and apoptotic pathways¹⁹. Specifically, caspases are significant targets of intracellular ROS that modulate apoptotic signaling cascades²⁰. ROS can affect signaling pathways that promote caspase-3 activation through reactive cysteine residue oxidation in target proteins, thereby altering protein-protein interactions and enzymatic activities. During autophagy induction, the C-terminal fragment of Beclin-1 localized to the mitochondria can sensitize cells to apoptosis, leading to enhanced caspase-3 activation²¹. The interplay between autophagy and apoptosis is characterized by the mutually regulatory effects of various signaling molecules. For instance, Bcl-2 modulates autophagic and apoptotic processes through its regulatory influence on proteins such as Beclin1 and the interactions between BAX and BAK dimers. Additionally, cellular FLICE-inhibitory protein (c-FLIP) inhibits caspase-8 activation and restricts autophagosome formation related to LC3 conjugation. Under heightened autophagic flux, Fas ligand (FasL) promotes the degradation of Fas-associated phosphatase 1 (Fap1), facilitating Fas activation and the execution of mitochondria-independent apoptotic pathways. Moreover, autophagy contributes to the degradation of inhibitors of apoptosis proteins (IAPs) to induce apoptotic processes. Autophagosomes can enhance caspase-8 activation through ATG5, LC3, and p62 complex. The reciprocal antagonism between apoptosis and autophagy manifests through the proteolytic cleavage or degradation of respective regulatory proteins in an enzyme-dependent manner. Active caspase-8 subsequently initiates a cascade of events leading to the activation of downstream effector caspases²², including caspase-3²³.

Beyond these processes, autophagy also plays a significant role in modulating endogenous inflammasome activators and components, such as pro-IL-1β—IL-1β increases in cells with oxidative stress²⁴. Autophagy is a crucial regulator of IL-1β release, influencing transcription, processing, and secretion pathways²⁵. The lens, a transparent structure essential for visual function, suffers impaired functionality under oxidative stress, consequently affecting visual quality. The objective of the present study is to elucidate the induction of autophagy within the lenses of Wistar rats subjected to chlorpyrifos (CPF) exposure, as evidenced by increased levels of MDA, H2O2, p62, LC3-II, caspase-3, and IL-1β, alongside decreased levels of SOD and mTOR in their lens tissues.

MATERIALS AND METHODS:

Materials:

The chlorpyrifos utilized in this study are pro-analytical grade chlorpyrifos, which appear as white crystals. It was produced by GlpBio, USA, and sourced from PT. Okta Saintika Laboratory in Malang. The research was conducted at the Pharmacology Laboratory of the Faculty of Medicine at the University of Jember, involving 24 male Wistar rats. These rats were divided into two groups: a control group and a treatment group, the latter receiving chlorpyrifos at a dosage of 5 mg/kg body weight, dissolved in 0.9% NaCl with the addition of 5% Tween 20.

Methods:

The measurements of MDA, H2O2, and SOD were conducted utilizing the Enzymatic Colorimetric method. In contrast, p62, LC3-II, caspase-3, and IL-1β levels were quantified through the Enzyme-Linked Immunosorbent Assay (ELISA) technique. Additionally, the evaluation of mTOR was performed using the Western Blot method.

Data Analysis:

The data analysis utilized the following methods: 1. Descriptive statistics: This was employed to calculate the mean, standard deviation (SD), minimum, and maximum values. 2. Normality test: Shapiro-Wilk test was performed to evaluate the data distribution, due to the sample size being less than 50. A p-value greater than 0.05 indicates that the data is normally distributed, while a p-value less than 0.05 suggests that the data is not normally distributed. 3. Homogeneity Test: This was performed to evaluate the variability of the data through the Homogeneity of Variance test. The data is regarded as homogeneous if the p-value exceeds 0.05, and not homogeneous if the p-value is below 0.05. 4. Parametric Statistical Analysis: An independent t-test to identify significant differences between the two groups. Random sampling was employed for the tests.

RESULT:

mTOR level:

Figure 1 illustrates the outcomes of the western blot analysis conducted on mTOR protein isolated from the lens proteins of Rattus norvegicus of the Wistar strain across two experimental groups: the control group and the group treated with CPF. The protein isolation procedure results in an approximate yield of 20mg/mL of protein for both groups. The qualitative assessment of the western blot results indicates a marked decrease in mTOR expression in the CPF-treated group (column 2) compared to the control group (column 1), suggesting an effect of CPF treatment on mTOR levels.

Figure 1. Western blot results of mTOR expression.

Notes: 1. Control group 2. CPF-treated group 3. Protein ladder

Results of Variable Measurements

The measurements of ROS were analyzed based on the levels of MDA, H2O2, SOD, p62 expression, Interleukin (IL)-1β expression, LC3-II expression, caspase-3 expression, and mTOR expression. These results are described in Table 1 below.

This table-1 presents the mean, minimum, maximum, and standard deviation (SD) variables measured across control and treatment groups.

Normality and Homogeneity tests:

The results of the normality and homogeneity tests are presented in Table 2 below.

Table 1. Descriptive data of research variables (n=24)

Variable	Group	n	Mean	Min	Max	SD
ROS	Control	12	2.11	0.22	4.86	1.55
	Treatment	12	2.68	0.33	7.65	2.16
p62	Control	12	5.61	3.71	8.34	1.44
	Treatment	12	14.09	8.34	20.01	4.06
IL-1β	Control	12	5.05	3.1	11.48	2.22
	Treatment	12	8.96	1.49	14.01	3.93
SOD	Control	12	71.57	64.71	88.24	10.59
	Treatment	12	70.1	41.18	88.24	14.72
LC3-II	Control	12	48.72	38.73	63.03	7.58
	Treatment	12	88.65	10.2	124.29	31.47
Caspase 3	Control	12	7.16	4.47	9.93	1.9
	Treatment	12	11.52	9.26	14.76	1.72
mTOR	Control	12	27.64	18.81	36.48	6.2
	Treatment	12	13.66	9.38	22.97	4.52
H2O2	Control	12	11.54	8.31	15.26	2.23
	Treatment	12	12.99	10.2	18.0	2.27

Table 2. Results of normality and homogeneity tests of variables

Variable	n	Normality Test (p)*	Description	Homogeneity Test (p)**	Description
ROS	24	0.027	Not Normal	0.563	Homogeneous
p62	24	0.014	Not Normal	0.000	Not Homogeneous
IL-1β	24	0.039	Not Normal	0.028	Not Homogeneous
SOD	24	0.146	Normal	0.298	Homogeneous
LC3-II	24	0.123	Normal	0.006	Not Homogeneous
Caspase-3	24	0.645	Normal	0.502	Homogeneous
mTOR	24	0.045	Not Normal	0.069	Homogeneous
H2O2	24	0.712	Normal	0.710	Homogeneous

p*= The p-value obtained from the Shapiro-Wilk test for normality. A p-value exceeding 0.05 signifies that the data follows a normal distribution, whereas a p-value below 0.05 indicates a deviation from normality. p**= The p-value resulting from the Homogeneity of Variance test. A p-value greater than 0.05 suggests that the data is homogeneous, while a p-value less than 0.05 reflects heterogeneous data.

Based on these results, variables such as ROS, p62, IL-1β, LC3-II, and mTOR do not follow a normal distribution, while variables like SOD, Caspase-3, and H2O2 show a normal distribution. Additionally, the data for some variables (e.g., p62, IL-1β, and LC3-II) are not homogeneous.

Comparison test (t-test):

Table 3. Results of comparison tests between groups

Variable	Group	n	Mean	SD	T-test (p-value)
ROS	Control	12	2.11	1.55	0.485
	Treatment	12	2.68	2.16
p62	Control	12	5.61	1.44	0.000
	Treatment	12	14.09	4.06
IL-1β	Control	12	5.05	2.22	0.012
	Treatment	12	8.96	3.93
SOD	Control	12	71.57	10.59	0.781
	Treatment	12	70.1	14.72
LC3-II	Control	12	48.72	7.58	0.001
	Treatment	12	88.65	31.47
Caspase-3	Control	12	7.16	1.9	0.000
	Treatment	12	11.52	1.72
mTOR	Control	12	27.64	6.2	0.000
	Treatment	12	13.66	4.52
H2O2	Control	12	11.54	2.23	0.128
	Treatment	12	12.99	2.27

p-value of less than 0.05 indicates a statistically significant difference between the groups. The p-value greater than 0.05 indicates no significant difference.

DISCUSSION:

The eye lens's structural integrity and physiological function affect visual quality preservation. Chlorpyrifos (CPF) administered orally exhibits an absorption rate exceeding 70%, whereas dermal administration results in less than 3% absorption. Following absorption, CPF is rapidly metabolized and excreted, with a half-life of approximately 27 hours in humans, predominantly via renal excretion as TCPy. The mechanism of toxicity associated with CPF is primarily attributed to its irreversible inhibition of acetylcholinesterase (AChE) activity in target tissues, resulting in the accumulation of acetylcholine (ACh), which can disrupt both somatic and autonomic nervous system functions. In the early stages of biotransformation, CPF undergoes desulfurization into chlorpyrifos oxon (CPO), a process facilitated by cytochrome P-450 enzymes. The formation of an irreversible bond between the sulfur atom of CPF and cytochrome P-450 catalyzes this reaction, leading to the generation of CPF oxon, which is recognized as the principal and most toxic metabolite responsible for AChE inhibition. This disrupts the equilibrium between oxidants and antioxidants, culminating in an increase in ROS²⁶. The lipophilic properties of CPF facilitate its penetration across the blood-ocular barrier, allowing both CPF and CPO present in the aqueous and vitreous humor to be gradually absorbed by the lens, particularly into the lens cortex²⁷.

Chronic exposure to CPF has been linked to ROS elevation production, resulting in oxidative stress. Autophagy, a critical self-regulatory process, removes oxidized cellular components and modulates intracellular ROS levels. ROS influences autophagic activity through both transcriptional and post-translational mechanisms. While ROS can initiate autophagy to maintain redox homeostasis and facilitate the degradation of oxidized organelles and cellular components, they may also inhibit autophagy by oxidizing autophagy-related proteins (such as ATG, ATG7, and ATG10) or by inactivating autophagy regulators including TFEB and Phosphatase and tensin homolog deleted on chromosome ten (PTEN)²⁸. Malondialdehyde (MDA), a byproduct of lipid peroxidation, is frequently utilized as a biomarker for oxidative stress²⁹. Exposure to the organophosphate pesticide CPF is associated with a significant increase in MDA levels²⁶. This study demonstrates a notable elevation in ROS levels, as indicated by MDA content,-when comparing control and treatment groups; however, the differences observed were statistically insignificant. Such findings may suggest an adaptive response of the body's endogenous antioxidant system as it mitigates the accumulation of ROS³⁰.

Superoxide dismutase (SOD) constitutes a pivotal metalloenzyme that plays an essential role in the antioxidant defense against oxidative stress within biological systems³¹. SOD catalyzes the dismutation of superoxide anion radicals (•O2−) into hydrogen peroxide (H₂O₂) and molecular oxygen (O2)³². Recent findings indicate that CPF diminishes both the expression and activity of SOD enzymes³³. Moreover, CPF has been shown to elevate levels of ROS, which have the potential to oxidize the active sites of proteins, consequently leading to a compromise in both functional capacity and enzyme activity³⁴. In this study, it was observed that the mean SOD enzyme activity in the treatment cohort was lower than that in the control group; however, this difference was not statistically significant.

The role of mitochondrial H₂O₂ in cellular signaling is critical, as H₂O₂ exhibits greater stability than other ROS molecules and can diffuse readily into the cytosol. This molecule is integral to several cellular signaling pathways to inactivate certain vital enzymes through the thiol group's oxidation at their active sites. Furthermore, H₂O₂ can permeate the cell membrane and engage in reactions with transition metals such as iron (Fe) and copper (Cu), leading to the formation of hydroxyl radicals (-OH) via the Fenton or Haber-Weiss reactions. Among reactive oxygen species, H₂O₂ represents the most stable oxygen species found in the aqueous humor, facilitating diffusion into the anterior lens³⁴. The study by Imam et al. (2018) stated that exposure to chlorpyrifos can cause oxidative stress, although it is not statistically significant³⁵. The H2O2 levels in this study were higher in the treatment group compared to the control group, although not statistically significant, indicating the potential for oxidative stress that may damage the lens tissue. The absence of references regarding the effect of chlorpyrifos exposure on lens H₂O₂ allows for further exploration in the future.

The mechanistic target of rapamycin (mTOR) is essential in orchestrating autophagic flux, encompassing phagophore and autophagosome formation, autolysosomal degradation, and the reformation of autophagic lysosomes. Autophagy is a cellular response to stress, often triggered by nutrient scarcity, particularly amino acids. This process is intricately regulated through signaling cascades such as mTOR and AMP-activated protein kinase (AMPK), a pivotal pathway in assessing cellular nutritional status³⁶. This study revealed a statistically significant disparity in mTOR expression between the control and treatment groups (p-value=0.000), indicating that CPF-induced oxidative stress affects mTOR level reduction. Notably, mTOR complex 1(mTORC1) inhibits autophagy by phosphorylating the Regulatory-associated protein of mTOR (RAPTOR) at Ser863. A study conducted by Hu et al. (2020) elucidated that the regulation of autophagy by leucine through mTOR acetylation is associated with the inhibition of mTOR translocation to the lysosome, concurrently leading to increased levels of LC3-II, which is a marker for enhanced autophagic activity³⁷.

The p62 protein functions as a selective autophagy adaptor, facilitating the aggregation with NF-E2-related factor 2-Kelch-like ECH-Associated protein 1 (NRF2/KEAP1). This interaction enhances the dissociation of the NRF2/KEAP1 complex, thereby promoting the translocation of NRF2 to the nucleus³⁸. Given that p62 is predominantly degraded through autophagy, its accumulation within cellular contexts denotes defective autophagic processes. Consequently, p62 has emerged as a crucial biomarker for autophagy inhibition and abnormalities associated with autophagic degradation³⁹. Recent investigations reveal a significant elevation in p62 expression in ocular lenses subjected to oxidative stress, particularly following CPF exposure. p62 serves as a vital indicator of autophagic activity, and within the framework of stress-induced autophagy, it plays a pivotal role in modulating the NFE2L2/NRF2-mediated antioxidant response pathway via interactions with KEAP1-binding domains³⁷. Furthermore, a notable reduction in LC3-II levels has been documented in the experimental group compared to controls, suggesting that CPF exposure disrupts autophagy in lens cells.

Elevated levels of reactive oxygen species (ROS) precipitate a redox imbalance that adversely affects the autophagic process directly and indirectly. The increase in ROS is linked to enhanced superoxide dismutase (SOD) activity⁴⁰, which facilitates the formation of hydrogen peroxide (H2O2), a significant oxidant implicated in cataractogenesis⁴¹. This condition concomitantly reduces the reduced form of ATG4, altering LC3B-I to LC3B-II via thiol modifications of Cys81 within ATG4, thus promoting autophagosome formation. The identification of the autophagic machinery has significantly advanced autophagy detection through various biochemical assays and LC3-based microscopy techniques, alongside experimental manipulation of autophagic pathways. Agents that influence autophagosome formation or subsequent degradation steps can manipulate autophagy pathways. An increase in LC3-II levels indicates heightened autophagosome presence⁴², thereby suggesting an impairment of the autophagic process in the lens.

Autophagy plays a critical role in modulating the production, processing, and secretion of interleukin-1 beta (IL-1β) through multiple mechanisms. It regulates IL-1β synthesis by targeting pro-IL-1β for lysosomal degradation and modulating NOD-like receptor protein 3 (NLRP3) inflammasome activation⁴³. Prior studies have demonstrated that pro-IL-1β is an autophagy target induced by rapamycin in macrophages⁴⁴. The findings of this investigation are consistent with previous literature, indicating that disruption of autophagic processes leads to increased IL-1β secretion. Elevated ROS levels serve as a critical signaling entity for NLRP3 inflammasome activation, prompting the release of pro-inflammatory cytokines such as IL-1β⁴⁵. The secretion of IL-1β initiates a cascade of inflammatory responses in lens cells, resulting in calcium pump dysfunction and further ROS production⁴⁶.

Moreover, this study reveals a statistically significant elevation in caspase-3 levels within the treatment group compared to controls, indicating that CPF exposure substantially influences caspase-3 activity in lens cells. Similar stressors can activate both the apoptosis and autophagy pathways simultaneously, and the interaction of key proteins in these pathways uncovers the molecular mechanisms that explain their connection. Evidence suggests that certain molecules can modulate autophagy and apoptosis, functioning as molecular switches that respond to cellular damage signals⁴⁷. Caspase-3, in conjunction with c-Jun N-terminal kinase (JNK) signaling, generates mitogenic signals that promote proliferation in adjacent cells via apoptosis-induced proliferation (AiP), where activated caspase-3 initiates apoptotic processes⁴⁸. Additionally, LC3 has been recognized as a platform facilitating apoptosis through caspase-8 activation mediation. Impaired degradation of autophagic proteins results in the accumulation of LC3 and SQSTM1/p62, leading to the aggregation and autoactivation of caspase-8, ultimately driving the apoptotic response⁴⁹.

CONCLUSION:

This study demonstrates that exposure to the organophosphate CPF affects autophagy in the lenses of Wistar rats, as indicated by reduced levels of mTOR. Moreover, the findings show that elevated levels of p62 and LC3-II are crucial autophagy markers. Oxidative stress resulting from CPF exposure is evidenced by increased levels of MDA and H2O2, alongside a decrease in superoxide dismutase (SOD) levels, further signaling oxidative stress. Additionally, elevated caspase-3 levels indicate apoptosis in the lens, corroborated by increased levels of the pro-inflammatory cytokine IL-1β, which may also be induced by p62. A key finding of this study is that, despite the statistically insignificant increases in oxidative stress markers, a significant induction of autophagy was observed.

This suggests that CPF may activate autophagy through alternative pathways. Given the continued widespread use of organophosphate pesticides and the identification of autophagy induction as a vital mechanism for maintaining cellular homeostasis, further research into these autophagy processes is warranted.

CONFLICT OF INTEREST:

The authors declare that they have no competing interests related to this research.

ACKNOWLEDGMENTS:

The authors express their gratitude for the financial assistance provided by the University of Jember project grant.

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Received on 07.01.2025 Revised on 05.05.2025

Accepted on 12.08.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):19-26.

DOI: 10.52711/0974-360X.2026.00003

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