Carbofuran pesticide toxicity to the eye

Abstract

Pesticide exposure to eyes is a major source of ocular morbidities in adults and children all over the world. Carbofuran (CF), N-methyl carbamate, pesticide is most widely used as an insecticide, nematicide, and acaricide in agriculture, forestry, and gardening. Contact or ingestion of carbofuran causes high morbidity and mortality in humans and pets. Pesticides are absorbed in the eye faster than other organs of the body and damage ocular tissues very quickly. Carbofuran exposure to eye causes blurred vision, pain, loss of coordination, anti-cholinesterase activities, weakness, sweating, nausea and vomiting, abdominal pain, endocrine, reproductive, and cytotoxic effects in humans depending on amount and duration of exposure. Pesticide exposure to eye injures cornea, conjunctiva, lens, retina, and optic nerve and leads to abnormal ocular movement and vision impairment. Additionally, anticholinesterase pesticides like carbofuran are known to cause salivation, lacrimation, urination, and defecation (SLUD). Carbofuran and its two major metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) are reversible inhibitors of acetylcholinesterase (AChE) which regulates acetylcholine (ACh), a neurohumoral chemical that plays an important role in corneal wound healing. The corneal epithelium contains high levels of ACh whose accumulation by AChE inhibition after CF exposure overstimulates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). Hyper stimulation mAChRs in the eye causes miosis (excessive constriction of the pupil), dacryorrhea (excessive flow of tears), or chromodacryorrhea (red tears). Recent studies reported alteration of autophagy mechanism in human cornea in vitro and ex vivo post carbofuran exposure. This review describes carbofuran toxicity to the eye with special emphasis to corneal morbidities and blindness.

1. Introduction

Pesticides are widely used in agriculture, horticulture, forestry, gardens, offices and for public and animal health (Gupta, 2006; Gupta and Milatovic, 2012; Sharma and Sharma, 2012). Carbamate (CM) compounds are also used in veterinary medicine as ectoparasiticides and in human medicine for therapeutic interventions of Alzheimer’s disease, myasthenia gravis, glaucoma, and in prophylaxis of organophosphate (OP) nerve gas poisonings (Bajgar et al., 2015; Gupta, 2006; Stojiljković et al., 2020; Woltjer and Milatovic, 2006). The commonly used N-methyl carbamates (aldicarb, carbofuran, carbaryl, methomyl, oxamyl, pirimicarb, propoxur, etc.) are esters of carbamic acid. Carbofuran is a highly toxic chemical commonly used as an insecticide, nematicide and acaricide for agricultural, forestry, and industrial applications, and in accidental and malicious poisonings (Gupta et al., 2018; Khan et al., 2021; Lv et al., 2022; Pivariu et al., 2020; Umeda et al., 2018).

Approximately 5 million pounds of carbofuran is utilized every year in the United States alone, and as a result, soil and water are contaminated significantly. About 45% of urban African American women show a trace amount of carbofuran in their plasma (Bonner et al., 2005). Nitrosated carbofuran shows mutagenic properties (Bonner et al., 2005). The United States Environmental Protection Agency (US EPA) reported several pesticide poisoning incidences from multiple sources including state and federal agencies. These reports indicate that the workers got carbofuran (a) in the eye while spraying in the field, (b) in the face, (c) farmer spilled on hands while spraying, (d) exposed during unloading of carbofuran from truck, splashed in the eyes, (e) person applied carbofuran for the whole day becoming ill, (f) exposed during a spray break, (g) exposed when cleaning up spray (US EPA, 1997).

Carbamate compounds produce toxicity in mammals, birds, fish, and wildlife primarily due to carbamylation (reversible inhibition) of acetylcholinesterase (AChE) and accumulation of ACh) at the synapses in the brain and the neuromuscular junctions in skeletal muscles (Gupta and Milatovic, 2012; Gupta et al., 2018; Mishra et al., 2020; Silberman and Taylor, 2022). This leads to the toxic signs of hypercholinergic preponderance. In humans and animals, carbofuran also exerts toxic effects due to non-cholinergic mechanisms, such as endocrine, reproductive, cytotoxic, genotoxic disorders, etc. (Goad et al., 2004; Gupta and Milatovic, 2012; Gupta et al., 2007; Mishra et al., 2020). In mammals, carbofuran exposure induces apoptosis of the neurons in the hippocampus, oxidative stress, neuroinflammation, memory dysfunction, and chromosomal abnormalities (Gupta and Milatovic, 2012; Gupta et al., 2007; Khan et al., 2021). A recent study reported that carbofuran toxicity increases the cellular aging process and interferes with biological aging in animals with abnormal spns1 (Khan et al., 2021). This review describes carbofuran toxicity in general and ocular toxicity in particular.

2. Carbofuran chemistry

Chemically carbofuran (CAS number, 1563-66-2; EINECS number, 216-353-0; and RTECS number, FB9450000) is 2,3-dihydro-2,2-dimethyl-7-benzofuranol methylcarbamate; 2,2-dimethyl-2,3-dihydro-7-benzofuranyl-N-methylcarbamate; methyl carbamic acid 2,3-dihydro-2,2-dimethyl-7-benzofuranyl ester; 2,2-dimethyl-7-coumaranyl-N-methylcarbamate; or 2,2-dimethyl-2,3-dihydro-1-benzofuran-7-yl methylcarbamate (IUPAC name). The chemical formula of carbofuran is C₁₂H₁₅NO₃ and molar mass is 221.256 g·mol⁻¹. Its structural formula is shown in Figure 1. Carbofuran is manufactured by the reaction of methyl isocyanate with 2,3-dihydro-2,2-dimethyl-7-hydroxybenzofuran. Some physical and chemical properties of carbofuran include white, crystalline solid, melting/point 150–152 °C, boiling point 313.3 °C, flash point °143.3C, specific gravity 1.18 at 20 °C, density 1.2 g/cm³, vapor pressure 2 × 10 ⁻⁵ mm Hg at 33 °C, water solubility 300 ppm at room temperature and 700 ppm at 25 °C, and octanol/water partition coefficient as log Pow 2.32. Carbofuran is highly soluble in N-methyl-2-pyrrolidone, dimethylformamide, dimethyl sulfoxide, acetone, acetonitrile, methylene chloride, cyclohexanone, benzene, and xylene. Evaporation of carbofuran at 20 °C is negligible.

Figure 1.

Chemical structure of carbofuran.

3. Carbofuran mode of action

3.1. Cholinergic mechanisms

Carbofuran, like other N-methylarbamate compounds, exerts acute intoxication in humans and animals by virtue of binding and inhibiting (carbamylation) acetylcholinesterase (AChE). The cholinesterases (ChEs) are serine hydrolases that normally catalyze the breakdown of acetylcholine (ACh), a neurohumoral transmitter. Carbofuran binds to the anionic subsite of AChE with high-affinity and makes a carbofuran-AChE complex. The rate of carbamylation is more imprtant than the binding of carbofuran to AChE. Potent AChE inhibition results from rapid carbamylation, as the carbamylation rate constant (k_i) is directly correlated with toxicity (Gupta, 1994). Sequestration of the AChE in carbamylated form thus precludes the hydrolysis of ACh, leading to excessive ACh accumulation. Severity of toxicity depends on the speed with which AChE is inhibited and the degree of AChE inhibition. AChE inhibition >70% in discrete brain regions (cortex, amygdala and hippocampus) and in diaphragm muscle leads to a toxic-level accumulation of ACh.

Accumulated ACh overstimulates ACh receptors in the brain, at neuromuscular junctions (NMJs) in skeletal muscles, and in other tissues. Muscarinic AChRs (mAChRs) and nicotinic AChRs (nAChRs) are functionally different. The mAChRs, which are G-protein coupled receptors, mediate a slow metabolic response via second messenger cascades. The nAChRs are ligand-gated ion channels, which mediate a fast-synaptic transmission of the neurotransmitter ACh. Skeletal muscles are enriched with nAChRs and are devoid of mAChRs. During AChE inhibition by OPs and CMs, unhydrolyzed ACh does not diffuse from the cleft, but repeatedly combines with post-synaptic receptors. The prolonged presence of ACh in the synaptic area appears to cause some of the myopathic changes. The common mAChR-associated toxicities include miosis, apprehension, hypersalivation, excessive tracheobronchial secretions, gastrointestinal cramps, broncho- and laryngospasms, dacryorrhea/chromodacryorrhea, urination, defecation, and bradycardia. The nAChR-associated toxicities are muscle fasciculations, tremors, convulsions, weakness of muscles, and seizures.

Evidence suggests that some CMs can directly interact with mAChRs and nAChRs and contribute to their toxicity (Gupta and Milatovic, 2012). In fact, CMs which have a more potent interaction with nAChRs are less potent inhibitors of AChE. The overstimulation of the somatic nervous system usually results in tremors, muscle fasciculations, and piloerection, as well as ataxia and paresis. Following an acute exposure, onset of toxicity signs/symptoms occurs within 10–15 min, maximal severity signs within 30–60 min, and death within one hour. Death ensues due to cardiac arrest and respiratory failure (Gupta, 1994; Gupta and Milatovic, 2012). The respiratory failure is of both central and peripheral origin.

During recovery period, methylcarbamic acid is removed from AChE and the enzyme is reactivated/regenerated by decarbamylation (Gupta, 1994; Timchalk, 2006). The rate of AChE decarbamylation varies among CM compounds. A slower the rate of decarbamylation results in slower recovery in humans and animals from toxicity. It is to note that the carbamylated enzyme half-life (t_½) for hydrolysis (~15–30 min) is substantially slower than for deacetylation (turnover time of ACh ~150 μsec). Carbamylated AChE from carbofuran does not ‘age’ as is reported with phosphorylated AChE (t_½ ~ days, irreversible inhibition) with OP pesticides or nerve agents (Gupta, 2006, 2020). Since the AChE recovers at almost the same rate as do the AChRs, a balance of AChE to AChRs is maintained over the postsynaptic surface during recovery. Therefore, a relatively constant ratio of AChE to AChRs is very important for maintaining normal neuromuscular function. Surviving humans and animals recover from toxic signs and symptoms of CMs within a few hours to 24 hours. However, the neuropsychological symptoms may persist for weeks or months (Fan, 2011).

3.2. Non-cholinergic mechanisms

In addition to cholinergic mechanisms, multiple non-cholinergic mechanisms are involved in the myriad of toxic effects by CMs/OPs (Gupta and Milatovic, 2012; Gupta et al., 2001a, b; Gupta et al., 2007; Milatovic et al., 2011; Milatovic et al., 2006). Such non-cholinergic targets include various enzymes, N-methyl-D-aspartate (NMDA) receptors, adenosinergic, γ-aminobutergic (GABAergic), and serotonergic systems, signaling molecules (such as nitric oxide) that are reported to be involved in seizures and lethality associated with anti-ChE pesticides and nerve agents (Dekundy and Kaminski, 2011; Gupta, 2020; Gupta and Milatovic, 2012; Gupta et al., 2007; Gupta et al., 2019; Lockridge and Schopfer, 2006). Carbofuran-induced unremitting excitotoxicity for an hour can cause neuronal degeneration in discrete brain areas such as cortex, amygdala, and hippocampus (areas primarily involved in initiation and propagation of convulsions and seizures) (Gupta et al., 2007). The early morphological alterations include dendritic swelling of pyramidal neurons in CA1 region of the hippocampus. The AChEI-induced neuronal cell death is a consequence of a cascade of extra- and intracellular events leading to the intracellular accumulation of Ca²⁺ ions and the generation of free radicals (Gupta et al., 2001a, b; Gupta et al., 2007). Excessive production of free radicals causes oxidative/nitrosative stress to which the brain is especially vulnerable. This leads to depletion of high-energy metabolites (ATP and phosphocreatine). Lipids are readily attacked by free radicals, resulting in the formation of a number of peroxidation products (formed non-enzymatically), such as F₂-isoprostanes, furans, and F₄-neuroprostanes (specific markers of oxidative stress to the neurons) (Gupta and Milatovic, 2012; Gupta et al., 2007; Zaja-Milatovic et al., 2009). The cholinergic and non-cholinergic mechanisms involved in CMs/OPs toxicity are depicted in Figure 2.

Figure 2.

Cholinergic and non-cholinergic mechanisms involved in CM/OP toxicity; Courtesy (Gupta and Milatovic, 2012).

3.3. Carbofuran-induced intermediate syndrome

Intermediate syndrome (IMS) was reported for the first time in Sri Lanka, where 10 human patients presented 24–96 hours after acute cholinergic crisis from exposure to OP pesticides, such as methamidophos, fenthion, dimethoate and monocrotophos (Senanayake and Karalliedde, 1987). These patients had acute muscle paralysis and some required ventilator support. Since then IMS has been diagnosed in many countries involving a dozen OPs. IMS appears to be due to insufficient oxime therapy and lack of oxygenation or ventilation. In 2005, carbofuran was also demonstrated to cause IMS in patients accidentally or intentionally exposed to large doses of this insecticide (Paul and Mannathukkaran, 2005). IMS is characterized by acute respiratory paresis and muscular weakness, primarily in the facial, neck and proximal limb muscles. In addition, it is often accompanied by generalized weakness, cranial nerve palsies, depressed deep tendon reflexes, ptosis and diplopia. These symptoms can last for days or weeks.

It has been suggested that the defect in IMS is at the neuromuscular endplate and postsynaptic level, but the effect of neural and central components in producing muscular weakness have not been ruled out. Evidently, some anti-AChE insecticides are greatly distributed to muscles and have a higher affinity for nAChRs. There is no specific antidote for IMS. For further detail on IMS, readers are referred to following research work (De Bleecker et al., 1993).

3.4. Carbofuran phamacokinetics and physiologically based pharmacokinetics

Pharmacokinetics of CM pesticides, including carbofuran, is concerned with the quantitative integration of absorption, distribution, metabolism, and excretion, and associated toxicological responses in several mammalian species (Gupta, 2006; Gupta et al., 1994; Timchalk, 2006). Carbofuran can be absorbed by the oral, inhalation and dermal routes or through eye contact. It is poorly absorbed through intact skin.

The metabolism of carbofuran has a significant impact on its overall toxicity. Following an oral ingestion, 75% of absorbed carbofuran is protein bound. Metabolism of free carbofuran appears to involve hydroxylation and/or oxidation reactions that result in the formation of carbofuran phenol, 3-hydroxycarbofuran, 3-hydroxycarbofuran-7-phenol, 3-ketofuran, and 3-ketofuran-7-phenol. The major metabolites are produced by hydroxylation at the benzylic carbon to give -3-hydroxycarbofuran, which is oxidized to the 3-ketocarbofuran when not blocked by formation of conjugates. The other researchers also identified with certainty: 3-hydroxycarbofuran, 3-ketocarbofuran, and their respective 7-hydroxy hydrolysis products (Metcalf et al., 1968). These phenols, together with carbofuran phenol, are present in the free state and have also been identified as conjugates in various biological systems. In in vitro studies, cytochrome P-450 2E1 is the major isoenzyme responsible for 3-hydroxycarbofuran formation (Usmani et al., 2004).

Several carbofuran metabolites (3-hydroxycarbofuran, 3-ketocarbofuran, 3-ketocarbofuran phenol, carbofuran phenol, and conjugate of 3-ketocarbofuran) in the urine of mice and rats orally dosed with radiolabeled carbofuran (Metcalf et al., 1968). Interestingly, oral exposure of rats to labeled [¹⁴C]carbofuran resulted in enterohepatic circulation of some metabolites, such as 3-hydroxycarbofuran (known to inhibit AChE) (Marshall and Dorough, 1979). Most metabolites of carbofuran form glucuronide or sulfate conjugates which are excreted in the urine. The bile contained predominantly 3-hydroxycarbofuran glucuronide, a metabolite having the carbamate ester linkage intact, which may be cleaved to yield a potent anti-AChE aglycone. Since enterohepatic cycling of glucuronides involves cleavage of the conjugate in the gut, biliary excretion may lead to increased systemic activity of toxic carbamate metabolites. The plasma elimination t_½ of 3-hydroxycarbofuran (64 min) is more than twice that of carbofuran (29 min). These findings clearly demonstrated that enterohepatic circulation may be a key factor in maintaining anti-AChE activity after carbofuran no longer exists in the body (Ferguson et al., 1984; Marshall and Dorough, 1979).

The majority of carbofuran metabolites (3-hydroxycarbofuran, 3-ketocarbofuran, and their conjugated products) are excreted in the urine, very little in the feces, and trace amounts in the milk (Gupta, 1994).

A PBPK/PD model for carbofuran in Sprague-Dawley rats, and the corresponding human model was derived by replacing the rat physiological structure with that of the human (Zhang et al., 2010; Zhang et al., 2007). The physiological structure included arterial blood, brain, skin, fat, GI tract, kidney, liver, lung, portal blood, and venous blood. A full GI compartment model including stomach, duodenum, lower small intestine, and colon was utilized to better understand carbofuran GI absorption, biliary circulation, and fecal elimination. The advantage of the application of the constructed PBPK/PD model enables the dose-response study and the toxicological endpoints to be estimated in silico, something that cannot be easily achieved by regular bench work. It needs to be mentioned that only when a well-calibrated and validated PBPK/PD model is constructed will these computerized study findings be useful.

Using a generic PBPK model and the IndusChemFate PBPK model, urinary concentration of 3-hydroxycarbofuran after exposure during an 8-hour shift to carbofuran at the level of TLV of 0.1 mg/m³, the model predicted concentration of free-3-hydroxycarbofuran at 0.005 μmol/L=1.1 μg/L. Some models can also evaluate the impact of their variability on model predictions and predict the inhibition of blood AChE and BuChE. PBPK/PD models suggest that there is no interaction among N-methylcarbamates (NMCs) nor is there competition from the NMCs with AChE. For further details on PBPK modeling of CM and OP pesticides, their interactions, and risk assessment from cumulative exposure (Lowit, 2006; Padilla, 2006; Timchalk, 2006).

4. Carbofuran and pesticide toxicity

Pesticides are used in the agricultural industry to protect and improve crops. However, agricultural use of pesticides can have unintended negative effects on the environmental conditions and humans (Sharma et al., 2012a). Although currently many countries banned carbofuran usage, carbofuran is still used as an insecticide in agriculture and households, affecting humans as well as animals because it contaminates the atmosphere, water bodies, and food products (Cinar et al., 2015). Carbofuran is anticholinesterase (inhibits cholinesterase) and metabolites 3-hydroxycarbofuran and 3-ketocarbofuran induce cholinergic and non-cholinergic biochemical, hematological, and immunologic toxicity (Gupta, 1994). Carbofuran and its metabolites can enter the placenta and initiate severe maternal-placental-fetal structural abnormalities (Gupta, 1994).

The rate of carbofuran adsorption and desorption in soils is determined by the type of clay and organic carbon ingredients, but carbofuran can easily contaminate groundwater from pesticide runoff due to its high solubility and high mobility in soils (Bermudez-Couso et al., 2011). Carbofuran runoff can also harm the environment since it induces acute toxic effects on freshwater algae and fish (Anton et al., 1993). Carbofuran pesticide can have a differential influence on microorganisms in the soil as well. Some pesticides influence the growth of microorganisms, but some show suppressive activity or no action on microorganisms. Carbofuran, however, increased the population of Azospirillum and other anaerobic nitrogen fixers in flooded and non-flooded soil conditions (Lo, 2010).

Carbofuran and pesticide exposures occur in occupational and non-occupational workers and has led to homicides and suicides (Sakunthala Tennakoon et al., 2013). Toxic pesticides can enter the body via the ocular route (Jaga and Dharmani, 2006). Repeated exposure to pesticides is a significant health risk to body, specifically to eyes. Aerial spraying of pesticides over plants also increases the risk of ocular exposure in people. Ocular pesticide contact is very common to humans and animals from pesticide manufacturing plant accidents (Jaga and Dharmani, 2006) or its intentional use in wars, conflicts, and terrorism.

Pesticide exposure can show mutagenic effects by inducing chromosomal abnormalities, deoxyribonucleic acid (DNA) damage, micronuclei formation or cytotoxicity in the cells. It has been reported that pesticide toxicity causes abnormal ocular movements and can also lead to ocular pathologies such as visual loss. Pesticide toxicity can cause Saku disease, an optico-autonomic peripheral neuropathy with myopia, astigmatism, vision defect, abnormal eye movement, optic neuronal inflammation, reduced plasma level of ChE, and neurological defects (Jaga and Dharmani, 2006). Increased cellular apoptosis in ocular tissue, especially in corneal cells, is common in pesticide toxicity (Sanyal and Law, 2019). Recent studies found alteration of autophagy mechanism in human cornea in vitro and ex vivo post carbofuran exposure (Kempuraj and Mohan, 2022). Chronic pesticide exposure causes defective cellular responses in the cornea leading to visual defects (Sanyal and Law, 2019). However, the ocular/corneal effects of carbofuran and pesticides in general are poorly understood. It has been our central postulate that carbofuran exposure to eyes causes corneal stromal pathologies and vision loss in humans and companion animals.

4.1. Carbofuran toxicity to non-ocular system

Exposure or ingestion of carbofuran in humans leads to multiple ocular and non-ocular morbidities and can be lethal if not treated immediately. People exposed to carbofuran while mixing with seeds, spraying pesticides, cleaning pesticide sprayers, etc. experience redness of hands, blurred vision, excessive constriction of the pupil, nausea, vomiting, headache, dizziness, tachycardia, tachypnea, salivation, high blood pressure, and fasciculation; and laboratory findings of hyperglycemia (Satar et al., 2005). Typical acute occupational carbofuran poisoning can cause immediate illness that quickly improves. These studies recommend that agricultural workers should be appropriately educated on the toxic effects of carbofuran exposure so that they can reduce exposure-associated toxicity (Satar et al., 2005).

Carbofuran readily pollutes air, food, water bodies, and causes significant ocular defects, vomiting, hypersalivation, diarrhea, respiratory abnormalities, and death (Nguyen et al., 2021). It suppresses the AChE enzyme in the central nervous system (CNS) and inhibits neuronal signals (Purushothaman and Kuttan, 2017). In physiological conditions, AChE inhibits neuronal signal transmission by hydrolyzing ACh in the CNS and peripheral nervous system (Colovic et al., 2013). Carbofuran shows toxic effects on blood vessels as evidenced by significantly reduced cell survival of human umbilical endothelium and upregulated intracellular reactive oxygen species (ROS) levels indicating increased oxidative stress, DNA damage, increased apoptosis, and defective vascular functions including the angiogenesis process (Saquib et al., 2021). Since carbofuran can cause toxic effects on the endothelial cells of the blood vessels, it could also affect corneal endothelium which plays an important in maintaining corneal hydration, homeostasis, and transparency.

The CF toxicity involves oxidative stress, cellular senescence and autophagy. Exposure to CF reduces nuclear erythroid 2-related factor (Nrf2) and glutathione S-transferase Pi isoform 1 (Gstp1) levels while influencing autophagy and senescence (Khan et al., 2021; Oda et al., 2022). CF exposure didn’t increase cancer risk in farmers (Bonner et al., 2005) but caused an adverse effect on cell membranes by altering oxidative balance and membrane stability (Sharma and Sharma, 2012). CF significantly elevated the levels of F₂-isoprostanes and F₄-neuroprostanes (in vivo biomarkers of lipid peroxidation and generation of ROS), and citrulline (a specific marker of nitric oxide (NO)/nitric oxide synthase (NOS) and reactive nitrogen species (RNS) (Gupta et al., 2007; Milatovic et al., 2006; Zaja-Milatovic et al., 2009). Carbofuran can induce aneuploidy or cell death with high doses, thereby reducing the fertilization and implantation process (Cinar et al., 2015; Shen et al., 2022).

4.2. Carbofuran toxicity to ocular system

Pesticides affect conjunctiva, cornea, lens, retina, and optic nerve, and cause abnormal ocular movement, excessive tearing, and loss of vision (Figure 3). Improved safety procedures and regulations can reduce pesticide-associated eye injury (Food Safety Commission of, 2019; Jaga and Dharmani, 2006).

Figure 3:

Diagrammatic representation of carbofuran toxicity to eye. Carbofuran morbidities to eye include corneal haze/scarring, miosis, lacrimation, dacryorrhea, chromodacryorrhea, cataract, conjunctivitis, retinal degeneration, and vision impairment.

Carbofuran functions as a cholinesterase inhibitor, thereby increasing the levels of ACh in the central and peripheral nervous systems. Ach can influence cell membrane permeability through pores (nicotinic receptors) (Ringvold and Reubsaet, 2016). CF ingestion is known to cause coma, respiratory failure from acute respiratory distress syndrome (ARDS), and cortical blindness from the increased ACh levels in the nervous system (Baban et al., 1998). Corneal epithelium possesses high ACh, but its role is not yet clearly known. Additionally, rabbit corneal epithelium is devoid of cholinergic receptors (Pesin and Candia, 1982). However, mammalian tissue studies report that the corneal epithelium contains the highest concentration of muscarinic receptors (Sloniecka and Danielson, 2020). The corneal epithelium contains large nerve endings but the level of ACh is relatively low compared to junctional tissues. The ACh could regulate water and ion transport into the corneal epithelium (Sloniecka and Danielson, 2020). The role of the cholinergic system on active ionic transport was analyzed using frog corneas. An exogenous ACh (2 mM) showed a moderate inhibition of sodium transport but had no effect on chloride transport. It suggested that only endogenous ACh could stimulate ionic transport but not the exogenous Ach (Pesin and Candia, 1982). Also, ACh influences wound healing in the cornea. ACh inhibited mRNA levels of collagen I, collagen III, collagen V, lumican, fibronectin, and alpha-smooth muscle actin (α-SMA) in quiescent keratocytes. Additionally, decreased gene expression of collagens (I, III, and V), lumican, fibronectin, and α-SMA were seen with ACh treatment during and after the development of fibrosis (Sloniecka and Danielson, 2020). This study suggested that ACh inhibited corneal fibroblasts to develop contractile structures by the activation of muscarinic ACh receptors and consequently fibrosis in corneal stroma (Sloniecka and Danielson, 2020). In another study, ascorbic acid in corneal epithelium is shown to decrease in response to ACh exposure (Ringvold and Reubsaet, 2016). ACh could increase the corneal re-epithelialization process by changing proliferation and apoptosis.

Corneal epithelial cells contain choline acetyltransferase (ChAT), AChE, and two muscarinic and nicotinic ACh receptors (mAChR) subtypes, and several nAChR subtypes. Continuous activation of corneal epithelial cells (CEC) by the muscarinic and nicotinic signaling pathways is important for CEC survival and migration. Additionally, this study exhibited that cholinergic stimulation of CEC increases expression of integrin and cadherin molecules that are involved in the re-epithelialization process (Chernyavsky et al., 2014).

TGF-β1 is a key cytokine in corneal fibrosis development and ACh modulates keratocyte conversion to myofibroblast through TGF-β1 in vitro (Sloniecka and Danielson, 2020). After an insult, TGF-β1 from the epithelium is poured into the stromal extracellular matrix (ECM) in an inactive TGF-β1 form. This inactive form is activated by integrins and proteases and initiates the wound healing process in the stroma. TGF-β1 provokes significant release of ECM components (collagens, fibronectin, etc.) and facilitates myofibroblasts formation which can be inhibited by the ACh (Mohan et al., 2022; Sloniecka and Danielson, 2020). ACh not only can regulate the initiation of corneal fibrosis but may decrease the level of a fibrotic condition from the previous injuries (Sloniecka and Danielson, 2020).

Autophagy regulates corneal function and vision. Our recent studies with human primary corneal fibroblasts and human cornea organ culture found that CF alters expression of autophagy signature genes Beclin 1 and microtubule-associated protein light chain 3 (LC3I)/II mRNA (Kempuraj & Mohan, 2022). This report first time indicated that CF is injurious to autophagy mechanism in the cornea. Furthermore, it suggested that CF exposure to eye, like alkali and warfare agents, could lead to vision impairing corneal pathologies in humans and animals (Fuchs et al., 2021; Gupta et al., 2020; Kamil and Mohan, 2021; Kempuraj and Mohan, 2022; Mohan et al., 2022; Sinha et al., 2021; Tripathi et al., 2020). Therefore, it is imperative to develop a better understanding of molecular mechanisms by which carbofuran and other pesticides compromise normal corneal function and vision.

4.3. Carbofuran toxicological studies in experimental animals

Pesticides enter into eye and damage tissues. Table-1 lists toxic effects of pesticides reported in humans and in various models. Increased pesticide poisoning is due to the increased rate of apoptosis, expression of death receptor (FAS/CD95), and caspase 3 and decreased cellular proliferation and improper wound healing in cornea and other ocular and non-ocular tissues (Ballantyne, 2006, 2009; Jaga and Dharmani, 2006; Sanyal and Law, 2019; Kontadakis et al., 2014). Rats exposed to CF showed a significant reduction in AChE, increase in ROS and RNS in cortex, amygdala, and hippocampus (Gupta et al., 2007), alteration in hypothalamus-hypophysial ovarian axis and hormonal imbalance (Baligar and Kaliwal, 2002), and and increased neuronal susceptibility to glutamate toxicity (Umeda et al., 2018). Also, CF reduced neurogenesis in the early stage of gestation, reduced the neuronal progenitor cells, increased neurodegeneration in the hippocampus, and induced cognitive disorders in rat offspring (Mishra et al., 2012). Acute carbofuran toxicity also included transient endocrine disruption in male rats (Goad et al., 2004). Interestingly, pretreatment of carboxylesterase inhibitor (iso-OMPA; 1 mg/kg, sc) 1 hour before CF exposure significantly mitigated CF’s toxicity (Gupta and Kadel, 1989).

Table 1:

Carbofuran (CF) toxicity and impact on human case reports, in vivo animals, and in vitro cell culture models.

Clinical studies
Subject	Exposure Route	Molecular Target	Therapeutic Interventions	Symptoms	Toxicity	Ref. No
Human	Oral Ingestion	Synaptic-release blockers of the acetylcholinesterase (AChE)	None	Epitaxis, Eyelid Hemorrhage, Hyphema, Congestion, Myocardial Infarct, Edema, Ecchymosis	Respiratory and Cardiac Depression	(Yen et al., 2015)
Human	Dermal, Absorption	None	None	Sleeping issues, Headache, lack of appetite	Mental Disorders	(Ong-Artborirak et al., 2022)
Human	Oral Ingestion	Blocking the activity of cholinesterase	Multi drug antibiotic therapy	Hypersalivation, Lacrimation, Pupil Constriction, Bronchoconstriction, Convulsions	Respiratory and Cardiac Arrest	(Klatka et al., 2021)
Human	Oral Ingestion	None	Atropine, Dopamine, dobutamine	Seizures	Respiratory failure,	(Lamb et al., 2016)
Human	Oral Ingestion	Blocking the activity of cholinesterase	Calcium channel blocking drugs and magnesium sulfate	Paresthesia	Sensorimotor Neuropathy	(Yang et al., 2000)
Human	Inhalation	None	None	Weakness, Fatigue, Cephalalgia, Disorientation, Abdominal pain, Vomiting	Genotoxicity	(Zeljezic et al., 2008)
Human	Oral Ingestion	None	None	Coma, Acute respiratory distress syndrome (ARDS), and Cortical blindness	Respiratory failure	(Baban et al., 1998)
Human	Dermal, Oral, Inhalation	None	None	Mutagenic	Lung Cancer	(Bonner et al., 2005)
Animal studies
Subject	Exposure Route	Molecular Target	Therapeutic Interventions	Symptoms	Toxicity	Ref. No
Dogs	Oral Ingestion	Synaptic-release blockers of the acetylcholinesterase	None	Epitaxis, Eyelid Hemorrhage, Hyphema, Congestion, Myocardial Infarct, Edema, Ecchymosis	Respiratory and Cardiac Depression	(Pivariu et al., 2020)
Mice and Rats	Oral Ingestion and IV	Hormonal imbalance, Acetylcholinesterase inhibition, Increase oxidative and LPO stress,	Bridelia tomentosa leaf extract, Curcumin, Kebar grass extract, Curcuma longa	Loss of Body Weight, Loss of liver weight, increased oxidative stress, changes in blood indices, effected estrous cycle and follicles	Hepatic Damage, Neurobehavioral disorders, liver, heart, and brain damage, neuronal vulnerability, dermal toxicity, Reproductive toxicity	(Baligar and Kaliwal, 2002; Ferguson et al., 1984; Gammon et al., 2012; Hossen et al., 2017; Jaiswal et al., 2014; Mondal et al., 2021; Purushothaman and Kuttan, 2017; Rai et al., 2009; Seth et al., 2019; Umeda et al., 2018; Yulitasari et al., 2021)
Fishes	Absorption and Ingestion	Acetylcholinesterase inhibition	Lycopene	Increased oxidative stress, Testicular lesions, cardiac edema	Behavioral and Histopathological alterations, reproductive toxicity, Cardiotoxicity	(Anton et al., 1993; Bretaud et al., 2000; Covert et al., 2020; Hamed and Osman, 2017; Oda et al., 2022; Saputra et al., 2021; Singh et al., 2003)
Cell culture studies
Subject	Tested Dose	Molecular Target	Therapeutic Interventions	Symptoms	Toxicity	Ref. No
Human umbilical vein endothelial cells	1000 μM	-	-	-	Intracellular ROS, Mitochondrial Membrane Potential, DNA Damage	(Saquib et al., 2021)
Human peripheral blood lymphocyte	5–100 μM	-	Vitamin C and E	-	DNA Damage and cytogenotoxicity	(Das et al., 2007; Naravaneni and Jamil, 2005; Sharma et al., 2012a; Sharma et al., 2012b; Sharma and Sharma, 2012)
Bacteria (Sinorhizobium Saheli)	25–100 μg/mL	-	-	-	Cellular Viability, Cellular Proliferation, Cellular Morphology, Biofilms	(Shahid et al., 2021)
Plant (Vigna mungo L).	25–100 μg/mL	-	-	-	Growth, morphology, survival, cellular respiration, and inner membrane permeability	(Shahid et al., 2021)
Human liver microsomes	2.5–300 μM	-	Recombinant cytochrome P450 enzymes	-	-	(Abass et al., 2022)
Mouse oocytes	10–400 μM	-	-	-	Cell death, Reduced fertilization rates	(Cinar et al., 2015)
Hamster ovary cells (CHOK1)	5–100 μg/mL	-	-	-	Genotoxicity and Cytotoxicity	(Soloneski et al., 2008)
Cat fibroblast cells	0.045–1.08 mM	-	-	-	DNA Damage and cytogenotoxicity	(Chandrakar T. R, 2020)
Bull squamous carcinoma cells	400 ppm	-	-	-	Cellular toxicity on cancer cells	(Amanullah and Hari, 2011)

5. Carbofuran and pesticide toxicity to civilians, military personnel, and war Veterans

Symptoms of CF toxicity can appear within five to forty-five minutes depending on the amount and location of exposure. These include miosis, bronchospasm, lacrimation, defecation, bronchorrhea, urination, defecation, emesis, and salivation (Nguyen et al., 2021). The underlying mechanisms involves, but not limited to, increase in parasympathetic activation/accumulation of ACh in the central and peripheral nervous system due inhibition of AChE. Further, CF exposure causes a mix of parasympathetic and few adrenergic symptoms of tachycardia, hypertension, and mydriasis, however, parasympathetic symptoms generally prevail (Satar et al., 2005; Silberman and Taylor, 2022). CF can also cause neurotoxicity by crossing the blood-brain barrier (Gupta et al., 2007).

The ocular toxicities of anti-AChE pesticides and chemical warfare agents in humans and animals are documented (Ballantyne, 2006, 2009; McNutt and Hamilton, 2015; McNutt et al., 2020). Miosis is a classical sign of CF toxicity, and it degree can be dose-related. Iritis can occur from CF and is generally associated with conjunctival hyperemia. Application of physostigmine, neostigmine, or diisopropylphosphorofluoridate (DFP) to rabbit eye initially causes hyperemia of the iris, increase in intraocular pressure and increase in capillary permeability, permitting entry of proteins that may cause an aqueous humor flare (Ballantyne, 2006, 2009). The pig, rabbit, and human lens treated with paraoxon results in 50% inhibition of oxygen consumption (Ballantyne, 2006, 2009; Gehring and Smith, 1971; Gupta, 2015; Santolucito and Whitcomb, 1971). Recently, in an intentional CF poisoning in dogs led to 3^rd eyelid/conjunctival hemorrhage, diffuse uveal congestion and unilateral or bilateral hyphema (Pivariu et al., 2020). Biomarkers of CF’s ocular toxicity may aid early detection, severity, and progression of CF-induced ailment, as well as can be used predict response to treatment (Kontadakis et al., 2014). High pesticide exposure causes ocular irritation in 99% subjects (Jaga and Dharmani, 2006). It led to degeneration of ciliary body, optic nerve, retina, and extraocular muscles. Clinically, most observed symptoms of ocular pesticide exposure include corneal opacity, conjunctivitis, and conjunctival chemosis/hyperemia (Jaga and Dharmani, 2006). Clinical eye symptoms post CF include pinpoint pupils, blurred vision, and sore eyes (United States Environmental Protection Agency, 1997).

Gulf War Illness (GWI) includes a neuroimmune disorder with chronic multisymptomatic ailments such as fatigue, myalgic encephalomyelitis/chronic fatigue syndrome, headaches, pain, sleep disorder and memory issues in the troops deployed to the Middle East in the Persian Gulf War (Operation Desert Storm/Desert Shield) of 1990–91 (Baksh et al., 2021; Chester et al., 2019; Michalovicz et al., 2020; White et al., 2016). GWI veterans showed blurred/double vision, dry eye symptoms, photophobia, ocular pain, cognitive dysfunction, neuroinflammation, astrocyte and microglial activations, and increased proinflammatory cytokine levels (Attaluri et al., 2022). Although the exact causes of the GWI are poorly known, about 41,000 military personnel could have been over-exposed to pesticides and inhibitory effects against AChE (Chester et al., 2019). Exposure to AChE inhibitors (AChEIs) induces GWI symptoms, mitochondrial dysfunction, and oxidative stress in GWI veterans. Further, AChEIs can cause decreased AChE activity, neuronal degeneration, and neuronal death in animal models of GWI (Chester et al., 2019). The dry eye symptoms and retinal nerve fiber layer thinning could be a biomarker for GWI (Baksh et al., 2021; Sanchez et al., 2022).

Several human epidemiological and clinical studies ocular toxicity from exposure to anti-ChEs (Ballantyne, 2006, 2009). A ocular toxicity was reported in a pesticide manufacturing plant in workers exposed to CM pesticide methomyl (Morse et al., 1979). A cross-sectional study of fenitrothion sprayers in India found macular degeneration in 16% of workers (Misra et al., 1985). School children from Saku region of Japan demonstrated a 65% incidence of optic neuritis and/or chorioretinal atrophy (Oto, 1971).

Anti-AChEs can modify retinal physiology and function. The retina has at least five neurotransmitters (GABA, glycine, dopamine, idolamine, and ACh) and pathogenesis of retinal lesions by anti-AChEs appears complex (Ballantyne, 2006, 2009). Retinal detachment has been noted as a complication of the treatment of glaucoma with anti-AChEs. Histopathological studies demonstrated that diazinon and DFP induced necrotic lesions in retinal cells (Ballantyne, 2006). From a mechanistic standpoint, some studies have suggested that inhibition of retinal AChE may not be a major etiological factor in the causation of retinal toxicity by anti-ChEs (Atkinson et al., 1994). However, the possibility exists that OP/CM-induced retinal degeneration may occur if a substantial inhibition of cyclic guanosine monophosphate (cGMP) occurs apart from inhibition of retinal AChE.

6. Current therapeutics for pesticide/carbofuran toxicity

Presently, the first line of treatment for carbofuran exposed eyes is to immediately flush eyes with copious amounts of water and removing contact lenses (Silberman and Taylor, 2022). In large ingestions of CF, gastric lavage or activated charcoal can be considered. Additionally, if the patient has seizures, benzodiazepines may be given even though there is limited data on their efficacy in CF toxicity (Silberman and Taylor, 2022). Atropine can be given to the patient in the acute setting to stabilize the patient’s cardiorespiratory status and reduce the symptoms of CF toxicity since atropine competitively antagonizes acetylcholine at muscarinic receptors (Silberman and Taylor, 2022). Pretreatment of Atropine and memantine in rats protected animals from hypercholinergic activity such as seizures by blocking pathways associated with neuronal oxidative damage in CF toxicity (Gupta et al., 2007). In addition, rats that were given therapeutic atropine with memantine antagonizes the acute toxicity of CF more than atropine and memantine individually (Gupta and Kadel, 1989). If it is certain that a patient has CF toxicity, pralidoxime may be withheld since studies have shown that pralidoxime may cause complications of increased AChE inactivation after CF dissociates spontaneously (Silberman and Taylor, 2022). Several additional therapeutic evaluations are reported to treat CF-induced toxicity in animals or humans. Curcumin administration significantly prevented CF-induced neurobehavioral disorders, activities of acetylcholinesterase, lactate dehydrogenase, creatine kinase, gamma-glutamyl transferase, and mitochondrial enzyme in animals (Purushothaman and Kuttan, 2017). Vitamin C pretreatment prevented CF induced oxidative stress and erythrocyte fragility in rats (Rai et al., 2009). Likewise, Vitamin E pretreatment showed protective effects in CF-induced oxidative stress (Jaiswal et al., 2014). Turmeric is shown to inhibit CF toxicity by increasing antioxidant enzymes and decreasing lipid peroxidation (Hossen et al., 2017). In an environmental scope, methods to degrade carbofuran include using up-flow anaerobic sludge blankets, modified up-flow anaerobic sludge blankets with tube settlers, and solar-photo-Fenton treatment (Lopez-Alvarez et al., 2012; Madhubabu et al., 2007). We have evaluated various modalities to mitigate chemically induced corneal scars by promoting physiological repair and preventing pathological events (Gupta et al., 2021; Mohan et al., 2021; Mohan et al., 2011; Tripathi et al., 2020).

7. Conclusion

Carbofuran is a highly toxic carbamate pesticide that causes severe toxicity. Following exposure, CF rapidly enters in the eye. Ocular toxicity largely involves cholinergic mechanisms involving AChE inhibition followed by ACh accumulation and unremitting overstimulation of mAChRs and nAChRs. Early symptoms of cholinergic toxidrome include miosis, tearing, and loss of accommodation. The noncholinergic mechanisms include various transmitter systems, oxidative stress, neuroinflammation, apoptosis, autophagy, signaling pathways, etc. Toxic ocular effects unrelated to cholinergic system include dry eye symptoms, neuroimmune disorders, abnormal ocular movement, and blurred or loss of vision. Development of novel animal models and mechanistic understanding is warranted to counter CF toxicity

Highlights.

• Carbofuran causes multiple corneal/ocular pathologies and blindness in humans.

• Carbofuran exposure modulates wound healing and autophagy mechanism in the cornea.

• In the eye, miosis, dacryorrhea, chromodacryorrhea, ocular pain and irritation, and blurred vision are common post carbofuran exposure.