Paraquat dichloride toxicity in dogs: Biochemical mechanisms, oxidative stress pathways and diagnostic challenges

Introduction

Paraquat dichloride is a widely used, highly toxic, broad-spectrum -non selective contact herbicide and potent desiccant. It is among the most commonly used herbicides globally and is primarily applied for the control of broad leaved weeds and grasses in a wide range of agricultural settings, although it is less effective against deep rooted species. Its use has increased with the adoption of no till and minimum till agricultural practices, where it is promoted for rapid vegetation clearance as part of conservation agriculture systems [1,2] . Paraquat is marketed predominantly as the dichloride salt and is also available as a dimethyl sulfate formulation. Chemically, it belongs to the bipyridylium group of herbicides, a class of quaternary ammonium compounds collec W L Y H O \ N Q R Z Q D V ‡ T X D W V • [3] . Paraquat exerts its herbicidal action by disrupting photosynthetic electron transport and inducing membrane lipid peroxidation, leading to rapid cellular dehydration and plant death [4] . Paraquat is a fast acting herbicide that affects exposed green plant tissues, typically killing susceptible plants within one to three days of application [5] . The World Health Organization (WHO) classifies paraquat as a Class II, moderately hazardous pesticide however, it is widely regarded as one of the most dangerous herbicides still in common use due to its high acute toxicity [6] . The Pesticide Action Network (PAN) International designates paraquat as a highly hazardous [7] . Toxicological databases, including the Toxicological Data Network (ToxNet) and the Integrated Risk Information System (IRIS) of the United States Environmental Protection Agency (US EPA), classify paraquat as a probable human carcinogen (Group C) [8, 9] . Paraquat exposure has been associated with reproductive [10,11] . Farmers, agricultural laborers and residents living near treated areas are particularly vulnerable to occupational, environmental and accidental exposure. The compound can be absorbed through intact skin, with significantly increased absorption when the skin is abraded or damaged [12] . Acute poisoning may occur via dermal, ocular or inhalational routes and clinical signs may be delayed with fatal outcomes frequently result from progressive respiratory failure [13] . Localized dermatitis, ocular injury and epistaxis are commonly reported among exposed individuals, while chronic low level exposure has been associated with structural lung changes, chronic bronchitis and exertional dyspnoea [14,15] . Although paraquat is registered for use in more than 130 countries, at least 32 countries including all member states of the European Union have banned or severely restricted its use because of serious human health concerns [16] . In Switzerland, the home country of Syngenta, the principal manufacturer of paraquat, its use has been prohibited since 1989 owing to its high acute toxicity [17] . Several certification and labeling organizations, including Fairtrade International, the Forest Stewardship Council and the Rainforest Alliance, as well as multinational food corporations such as Dole and Chiquita and major retailers including Marks & Spencer, have voluntarily eliminated paraquat from their supply chains [18] . In India, paraquat dichloride 24% SL is registered with the Central Insecticides Board and Registration Committee (CIBRC) as the sole approved formulation and is categorized as highly toxic despite the absence of explicit CIBRC recommendations, its use has been approved for nine crops [19] . In contrast, paraquat dimethyl sulfate was banned in India in 1993. Paraquat dichloride continues to rank among the twenty most commonly recommended pesticides nationwide, although it is not the single most widely used herbicide [20] . Paraquat is frequently implicated in cases of deliberate selfpoisoning, particularly in agricultural regions. No specific antidote exists and the reported case fatality rate for paraquat poisoning ranges from approximately 42% to 80% [21, 22] . Global estimates suggest that tens of thousands of deaths annually may be attributable to paraquat ingestion [23] . In India, paraquat poisoning has been reported from northern, southern and northeastern states. In Kerala, the distribution, sale and use of paraquat were halted in 2011, along with sixteen other highly hazardous pesticides, due to severe health concerns; data obtained under the Right to Information Act indicate that paraquat is no longer in use in that state [24] . Despite its well recognized role in causing serious morbidity and mortality among agricultural workers and surrounding communities worldwide, systematically documented clinical and veterinary data on paraquat toxicity from India remain limited.

Case Presentation

A total of ten dogs (seven non-descript breeds, two Spitz and one German Shepherd) were presented to the Veterinary Clinical Complex, Veterinary College and Research Institute, Salem, over a six month period, 2-3 days after accidental ingestion or inhalation of paraquat. Respiratory distress was the most common complaint reported by all owners. All ten dogs tested positive for paraquat poisoning. The clinical signs varied among individual dogs, depending on the quantity of poison ingested, duration since exposure and immune status of the animal. In three cases, environmental investigations were conducted, which confirmed the presence of paraquat containing herbicides at the exposure sites. Three dogs recovered, while the remaining dogs succumbed to the condition despite treatment. Necropsy findings in deceased dogs revealed pulmonary haemorrhage, fibrosis and consolidation, along with hepatomegaly, splenomegaly and pneumomediastinum. Microscopically, inter and intra alveolar haemorrhage and pulmonary oedema were observed.

Diagnostic Techniques

The sodium dithionite test (fig. 1) is a rapid, simple, qualitative screening method used for the detection of paraquat in biological samples and is widely employed in both human and veterinary toxicology. The test is based on the reduction of paraquat, a bipyridylium compound, by sodium dithionite under alkaline conditions to form a stable blue coloured paraquat radical ion. Urine is the preferred sample due to higher sensitivity, although plasma, serum or gastric contents may also be used. Following alkalinisation of the sample, the addition of freshly prepared sodium dithionite results in the immediate development of a blue or blue purple colour in positive cases. The intensity of the colour roughly correlates with paraquat concentration, but the test remains qualitative in nature. A strongly positive reaction shortly after exposure supports the diagnosis and is often associated with a poor prognosis, whereas a negative result does not exclude paraquat toxicity in delayed presentations because paraquat is rapidly cleared from circulation and redistributed into tissues such as the lungs and skeletal muscle. Although the sodium dithionite test lacks the sensitivity and specificity of advanced analytical techniques such as high performance liquid chromatography or gas chromatography mass spectrometry, it remains a valuable bedside screening tool for early presumptive diagnosis of paraquat poisoning in dogs when interpreted in conjunction with clinical findings and exposure history.

H U L S K H

Electrocardiographic (ECG) evaluation in dogs (fig. 3,4) with paraquat intoxication consistently revealed sinus tachycardia as the predominant abnormality. This finding was primarily attributed to severe hypoxaemia, respiratory distress and heightened sympathetic stimulation secondary to progressive paraquat induced pulmonary injury. In some cases, mild nonspecific ST-segment and T-wave changes were observed, likely reflecting secondary myocardial hypoxia rather than primary cardiac disease, as no consistent arrhythmias or conduction disturbances were detected. These ECG alterations closely paralleled changes in Thoracic and abdominal radiographic examination (Fig. 5,6) in dogs with paraquat intoxication revealed characteristic changes consistent with severe pulmonary and soft tissue injury. Pneumomediastinum was identified by the presence of radiolucent streaks outlining the cardiac silhouette, trachea and major mediastinal structures, indicating the presence of free air within the mediastinum. This finding is suggestive of alveolar rupture secondary to severe pulmonary damage and increased intrathoracic pressure associated with dyspnoea and coughing. In some cases, air was observed tracking caudally from the mediastinum into the abdominal cavity, resulting in pneumoperitoneum (pneumo abdomen). The presence of free intraperitoneal gas was evident as abnormal radiolucency outlining abdominal organs and elevating the diaphragm. These radiographic findings reflect the severity of paraquat-induced pulmonary injury and are associated with a poor prognosis, as they indicate extensive alveolar destruction and air leakage along fascial planes.

Haematology

Ultrasound study

Paraquat intoxication in dogs causes marked oxidative damage to the lungs, particularly affecting the alveolar lining cells and pulmonary capillary endothelium. This results in acute lung inflammation, pulmonary oedema, haemorrhage and progressive scarring of the lung tissue.

Quantitative Confirmation

Paraquat can be quantitatively detected and confirmed in biological samples using several analytical techniques available in specialised laboratories. Spectrophotometric methods are commonly employed for rapid screening and estimation based on the characteristic absorption of paraquat, although their sensitivity and specificity are limited. High performance liquid chromatography (HPLC) offers improved accuracy and sensitivity and is widely used for quantitative determination of paraquat in blood, urine and tissue samples. Gas chromatography coupled with mass spectrometry (GC-MS) provides highly sensitive and specific identification and quantification, allowing definitive confirmation even at low concentrations. Radioimmunoassay (RIA) is another sensitive technique that enables detection of paraquat in biological matrices through antigen antibody reactions, though its availability is restricted to specialised facilities. These analytical methods play a crucial role in confirming exposure, assessing severity and supporting the diagnosis of paraquat intoxication in clinical and forensic investigations.

Mechanism of toxicity: Paraquat exerts its toxic effects in dogs primarily through the generation of reactive oxygen species (ROS), resulting in widespread oxidative stress and multi organ injury. Following ingestion, paraquat is rapidly absorbed from the gastrointestinal tract and distributed systemically, with preferential accumulation in the lungs via an active polyamine uptake system present in alveolar type I and type II pneumocytes. Within cells, paraquat undergoes redox cycling, in which it is reduced by nicotinamide adenine dinucleotide phosphate (NADPH) dependent enzymes to a paraquat radical that is rapidly reoxidized in the presence of molecular oxygen. This continuous redox cycling leads to excessive production of superoxide anions, hydrogen peroxide and hydroxyl radicals. The generated ROS cause lipid peroxidation of cellular and organelle membranes, oxidation of proteins and DNA damage, ultimately resulting in mitochondrial dysfunction, depletion of intracellular antioxidants such as glutathione and cell death by necrosis and apoptosis. In the lungs, oxidative injury to alveolar epithelium and pulmonary capillary endothelium leads to acute alveolitis, pulmonary oedema, haemorrhage and severe impairment of gas exchange. Surviving dogs often develop progressive interstitial fibrosis due to fibroblast activation and excessive collagen deposition, culminating in irreversible respiratory failure. Paraquat also induces significant extra pulmonary toxicity. Renal injury occurs due to direct tubular epithelial damage and ischemia, resulting in acute kidney injury that further impairs paraquat excretion and exacerbates systemic toxicity. Gastrointestinal effects include corrosive injury to the oral cavity, oesophagus and stomach, leading to ulceration, inflammation and haemorrhage. Hepatic injury results from oxidative hepatocellular damage, while central nervous system involvement may occur secondary to hypoxia and metabolic derangements. Collectively, the mechanism of paraquat toxicity in dogs is characterized by persistent oxidative stress organ specific accumulation particularly in the lungs and progressive, often fatal, multi organ failure in the absence of a specific antidote.

Biochemical pathway of paraquat toxicity

Mechanism of toxicity in lungs and muscle

Following ingestion, paraquat is rapidly absorbed and selectively taken up by the lungs (fig. 9) within the first few hours, achieving pulmonary concentrations several-fold higher than plasma levels. Within approximately 6 hours, paraquat induces acute alveolitis through intense oxidative injury to alveolar epithelial and capillary endothelial cells. By the third day post-exposure, profibroblasts migrate into the alveolar spaces, initiating fibroproliferative responses. By around day seven, progressive pulmonary fibrosis becomes evident, resulting in marked impairment of alveolar gas exchange. This culminates in severe hypoxaemia, tissue anoxia and death due to asphyxia in most affected dogs. In parallel, paraquat is also taken up by skeletal muscle, which acts as a secondary reservoir. Although plasma paraquat levels may decline and qualitative tests such as the sodium dithionite test may become negative, paraquat continues to be released slowly from muscle tissue (fig. 10) back into circulation. This sustained redistribution perpetuates pulmonary exposure and contributes to delayed worsening of lung injury and fatal outcomes. The prognostic relationship between plasma paraquat concentration and survival demonstrates a strong inverse correlation, with higher early plasma concentrations associated with extremely poor survival. Survival probability decreases sharply with increasing plasma levels during the initial hours after ingestion, while lower concentrations are associated with improved but still guarded outcomes. However, declining plasma concentrations do not necessarily indicate recovery, as continued release from tissue stores, particularly muscle, can maintain toxic pulmonary concentrations and drive progressive respiratory failure. Detailed post-mortem examination revealed severe multisystem lesions, with the lungs (fig. 11) showing the most prominent and diagnostically significant changes. The lungs were markedly congested, enlarged and heavy, failing to collapse upon opening of the thoracic cavity. The pleural surfaces were dark red to bluish-red and glistening, with multifocal petechial to ecchymotic haemorrhages. On sectioning, copious amounts of serosanguinous, frothy fluid exuded from the cut surfaces, confirming severe pulmonary oedema. The pulmonary parenchyma was firm and rubbery in consistency in several areas, indicating consolidation and early fibroproliferative changes consistent with paraquatinduced alveolar injury. The liver (fig. 12) was diffusely enlarged with rounded edges and a tense capsule. The hepatic parenchyma appeared dark, congested and friable, with accentuation of the lobular pattern on cut section. These changes are suggestive of toxic hepatocellular injury compounded by hypoxia and circulatory compromise. Mild bile staining was occasionally observed, although no focal necrosis was evident grossly. The spleen (fig. 13) was moderately to markedly enlarged and congested, with a dark red to purplish coloration. On cut section, the splenic pulp was soft and oozed blood, indicating vascular engorgement and systemic inflammatory response. Splenic enlargement was interpreted as secondary to acute circulatory disturbances and hypoxic injury. Overall, the necropsy findings were dominated by severe pulmonary congestion and oedema, accompanied by hepatomegaly and splenomegaly, reflecting the systemic nature of paraquat toxicity. The severity of pulmonary lesions correlated with the rapid clinical deterioration and fatal respiratory failure observed in affected dogs. Microscopic examination (fig. 14) of the lung revealed severe and diffuse pathological alterations consistent with acute toxic lung injury due to paraquat. The alveolar spaces were variably collapsed or distended and filled with eosinophilic proteinaceous oedema fluid, fibrin strands and inflammatory cell infiltrates. Marked congestion of alveolar capillaries and interalveolar septa was evident, along with multifocal haemorrhages. The alveolar septa were markedly thickened due to oedema, infiltration by neutrophils and macrophages and early fibroblast proliferation. Degeneration and necrosis of alveolar epithelial cells were observed, with desquamation of pneumocytes into the alveolar lumen. In some areas, bronchiolar epithelial damage with luminal cellular debris and inflammatory exudate was noted. These histological features are indicative of acute alveolitis progressing towards fibroproliferative changes, which correlate with paraquat induced oxidative injury and explain the severe impairment of gas exchange and fatal respiratory failure observed clinically.

Histopathology of Lung

Other Clinical Signs

In addition to respiratory compromise, dogs with paraquat intoxication commonly exhibit systemic and gastrointestinal manifestations. Cardiovascular involvement is reflected by an elevated heart rate, often secondary to hypoxaemia and systemic stress. Progressive multi-organ failure may occur as a consequence of widespread oxidative injury. Gastrointestinal signs such as vomiting and diarrhoea are frequent and are attributed to the corrosive effects of paraquat on the gastrointestinal mucosa. Laryngopharyngitis, excessive salivation and oral discomfort are also commonly observed due to direct chemical irritation. Respiratory distress remains the predominant clinical feature and progressively worsens despite therapeutic intervention.

Treatment

Therapeutic management of paraquat poisoning in dogs is largely supportive and aimed at limiting pulmonary inflammation, oxidative injury and secondary complications, as no specific antidote is available. Attempts may be made to reduce pulmonary inflammation and fibrosis through the administration of anti-inflammatory and immunosuppressive agents, as well as inhibitors of collagen synthesis. Corticosteroids such as methylprednisolone (0.5-1 mg/kg) or dexamethasone (0.1-0.2 mg/kg), alone or in combination with cyclophosphamide, have been used with the objective of attenuating fibroproliferative lung injury, although clinical efficacy remains inconsistent. Broad-spectrum antibiotics, including amoxicillin (10-20 mg/kg), enrofloxacin (4-7.5 mg/kg) and ceftiofur sodium (1-2 mg/kg), may be administered to prevent or treat secondary bacterial infections, particularly those involving the respiratory tract. Angiotensin-converting enzyme inhibitors such as enalapril and captopril have been reported to reduce paraquat-induced pulmonary fibrosis by modulating profibrotic pathways. Oxygen supplementation is contraindicated in paraquat poisoning, as increased oxygen tension enhances freeradical generation and exacerbates oxidative lung injury. Diuresis using agents such as furosemide (1-2 mg/kg) is recommended to reduce paraquat concentration within renal tubules and mitigate nephrotoxicity. Saline cathartics may aid in the rapid elimination of paraquat from the gastrointestinal tract if administered early. Extracorporeal techniques such as haemoperfusion and haemodialysis have limited or questionable benefit however, they may prolong survival if initiated shortly after ingestion. Fluid therapy is generally contraindicated due to the presence of severe respiratory distress and the risk of worsening pulmonary oedema. Induction of emesis and gastric lavage are also contraindicated because of the corrosive nature of paraquat and the potential for continued mucosal injury to the gastrointestinal tract.

Antioxidant Therapy

Given the central role of oxidative stress in paraquat toxicity, antioxidant therapy is commonly incorporated into treatment protocols. Vitamin E (300 mg orally twice daily) and vitamin C may be administered to reduce free-radicalmediated cellular damage. N-acetyl cysteine is used to replenish intracellular glutathione stores and may be administered at a loading dose of 150 mg/kg over 3 hours, followed by 300 mg/kg over 24 hours for up to three weeks. Desferrioxamine (100 mg/kg over 24 hours) acts as an iron chelator, thereby limiting iron-catalysed hydroxyl radical formation. Salicylic acid has also been suggested for its ability to scavenge hydroxyl radicals and inhibit activation of nuclear factor-% 1 ) -% D N H \ P H G L D W inflammation and fibrosis.

Conclusion

Paraquat toxicity should be considered an important differential diagnosis in dogs presenting with unexplained respiratory distress, gastrointestinal signs, renal dysfunction and a history suggestive of environmental exposure to paraquat. Given the relative ease of access to paraquatcontaining formulations such as Gramoxonefi 250 (Syngenta), the risk of intoxication in both humans and animals remains significant, whether accidental, intentional as in cases of self-harm or through malicious exposure. The absence of a proven and effective antidote in both human and veterinary medicine, combined with the rapid progression of severe pulmonary injury and irreversible fibrosis, renders therapeutic management extremely challenging and results in a generally poor prognosis. Nevertheless, experimental studies, particularly in rodent models, have demonstrated promising results with early intervention using high-dose parenteral lysine acetylsalicylate, various antioxidant therapies and selected angiotensin type I converting enzyme inhibitors, which may attenuate oxidative damage and limit progressive pulmonary fibrosis. Further clinical research is required to evaluate the translational potential of these therapies in canine paraquat intoxication.

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