Assessing the Risk of Epidemic Dropsy from Black Salve Use Short Title: Could Black Salve Cause Epidemic Dropsy? Andrew Croakera,B,C, Graham J

Liu Lei (Orcid ID: 0000-0002-3710-7042)

Mini-Review Full Title: Assessing the risk of epidemic dropsy from black salve use Short Title: Could black salve cause epidemic dropsy? Andrew Croakera,b,c, Graham J. Kinga, John H. Pyned, Shailendra Anoopkumar-Dukiec,e and Lei Liua*.

aSouthern Cross Plant Science, Southern Cross University, Lismore, NSW, Australia bWesley Medical Research Institute, Wesley Hospital, Auchenflower, QLD, Australia cQuality Use of Medicines Network, Queensland, Australia dSchool of Medicine, University of Queensland, St Lucia, QLD, Australia eSchool of Pharmacy and Pharmacology, Griffith University, Gold Coast Campus, Gold Coast, QLD, Australia

Standard Abstract Epidemic dropsy is a potentially life threatening condition resulting from the ingestion of argemone oil derived from the seeds of Argemone mexicana Linn. Exposure to argemone oil is usually inadvertent, arising from mustard cooking oil adulteration. Sanguinarine, an alkaloid present in argemone oil, has been postulated as a causative agent with the severity of epidemic dropsy correlating with plasma sanguinarine levels. Cases of epidemic dropsy have also been reported following the topical application of argemone containing massage oil. Black salve, a topical skin cancer therapy also contains sanguinarine, but at significantly higher concentrations than that reported for contaminated massage oil. Although not reported to date, a theoretical risk therefore exists of black salve inducing epidemic dropsy. This literature review explores the presentation and pathophysiology of epidemic dropsy and assesses the risk of it being induced by black salve.

Short Abstract Exposure to sanguinarine containing topical oils has been associated with the development of epidemic dropsy, a potentially fatal condition that can result in multisystem organ failure. Black salve, an alternative therapy used to treat skin malignancies, in some formulations contains sanguinarine in high concentrations. This suggests black salve use may place patients at risk of epidemic dropsy. This mini-review investigates the pathophysiological link between sanguinarine and epidemic dropsy, determining the potential for it to be caused by black salve therapies.

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jat.3619

This article is protected by copyright. All rights reserved. Introduction Black salve is a controversial, self-administered skin cancer treatment often purchased online. Having never been the subject of a clinical trial, its efficacy is currently unknown. A number of black salve toxicities have however been reported in case studies, including excessive scarring, disfigurement and disease recurrence resulting in death (Croaker et al., 2017a).

Sanguinaria canadensis, a core ingredient of black salve preparations, contains a number of biologically active alkaloids including the benzophenanthridine alkaloid sanguinarine (Figure 1) (Croaker et al., 2016). Concerns have been raised about the potential genotoxic and carcinogenic potential of sanguinarine (Croaker et al., 2017b), especially when applied to skin already damaged by ultraviolet radiation. Sanguinarine exposure has also been associated with an acute toxicity syndrome known as epidemic dropsy (Schneider et al., 2013) that typically results from the ingestion of oil derived from the seeds of Argemone mexicana Linn.

When assessing potential toxicities that may develop from the use of black salve therapies, it is useful to assess toxicities arising from plants that also produce S. canadensis alkaloids. S. canadensis, is a member of the Papaveracaea and produces benzophenanthridine alkaloids (BAs). These alkaloids have a limited phylogenetic distribution, being found in only three other plant families: the Caprifoliaceae or Honeysuckle family, Meliaceae or Mahogany family, and Rutaceae or Citrus family (Simanek, 1985). The incidence of BAs is primarily limited to the Papaveraceae and Rutaceae, with occurance in single genera of the Caprifoliaceae (Symphoricarpos) (Santavy, 1970) and Meliaceae (Xylocarpus). A. mexicana, originally a native of Mexico and the West Indies, has become a pantropical weed widespread in Africa and the Indian subcontinent. A. mexicana is also a member of the Papaveracaea and produces BAs.

A. mexicana seeds have been found to contain sanguinarine and dihydrosanguinarine (Figure 1) (Brahmachari et al., 2013). As the severity of epidemic dropsy appears to correlate with serum sanguinarine levels (Banerjee et al., 2000), it has been suggested that sanguinarine may have a pathogenic role in the development of this condition. With sanguinarine being a black salve constituent, there is therefore a theoretical risk that epidemic dropsy may develop from its use.

What is epidemic dropsy? Epidemic dropsy is a condition caused by the ingestion of edible oils adulterated with Argemone mexicana oil known as argemone oil (AO). Poisoning usually occurs in sporadic outbreaks where mustard oil is extensively used for cooking. Unscrupulous traders, adulterate

This article is protected by copyright. All rights reserved. mustard oil with cheap AO obtained from A. mexicana seeds (Ghosh et al., 2005). These seeds are dark in colour and of similar shape and size to mustard seeds from Brassica rapa (syn. campestris) and B. nigra (Gomber et al., 1994; Thatte & Dahanukar, 1999). The ready availability of argemone seeds and complete miscibility of the two oils make mustard oil adulteration a frequent occurrence that is difficult to detect (Sanyal, 1950). AO poisoning has also been reported following the ingestion of contaminated till oil, groundnut oil (Tandon et al., 1975) and contaminated ghee (Narasimhan et al., 1991). AO contains 0.6% w/v alkaloids consisting of 87% dihydrosanguinarine (DHSG), 5% sanguinarine (Figure 1), 0.57% berberine, 0.34% protopine, 0.12% chelerythrine and 0.03% coptisine (Upreti et al., 1991). Poisoning from AO has largely been attributed to the sanguinarine it contains (Sharma et al., 1999).

The earliest instance of epidemic dropsy was reported to have occurred in Calcutta in 1877 (Smith 1880) where it was called acute oedema. Since then several epidemics have occurred in India as well as in other countries such as Burma, Fiji, Madagascar and Mauritius (Manson-Bahr & Apted, 1982). The most extensive outbreak of epidemic dropsy in recent times occurred in New Delhi in 1998, resulting in over 3,000 hospitalizations and 65 deaths (Das & Khanna, 1998). Historically, higher fatalities have been reported in other occurrences with 1575 deaths reported in the 1926 Calcutta outbreak (Lal & Roy, 1937). In India the incidence of epidemic dropsy peaks between July and August when mustard oil, harvested toward the end of summer, is sold (Lal & Dasgupta, 1942). While the main method of exposure has been attributed to contaminated cooking oil ingestion, one epidemic in the North West Cape districts of South Africa occurred through A. mexicana contamination of wheat flour (Steyn, 1950). During a 2003 outbreak in Shivpuri and Sheopur, Madhya Pradesh state, that affected 480 people, sanguinarine levels were used as an index of mustard oil adulteration. Household mustard oil had estimated AO concentrations of 1.22-8.77% (Ghosh et al., 2005). Adulteration levels above 1% being sufficient to produce the clinical symptoms of epidemic dropsy (Sharma et al., 1999).

Epidemic dropsy has also been described in individuals following transcutaneous exposure to A. mexicana adulterated mustard oil. Four individuals, with a confirmed negative history of oral mustard oil intake, developed epidemic dropsy following 3.5-5% adulterated body massage oil exposure during a New Dehli outbreak in 1984 (Sood et al., 1985). As a daily routine, these individuals from different households had 20-40ml of mustard oil applied to their bodies for 30 to 60 minutes prior to bathing. The maximum massage oil sanguinarine concentration was reported at 0.0015%, these cases resulted in serum sanguinarine levels of 2-4 µg/100ml and urinary sanguinarine levels of 0.6-1.2 µg/100ml (Das & Khanna, 1997).

This article is protected by copyright. All rights reserved. The concentration of sanguinarine in black salve varies significantly between different vendor products. Some black salves contain a sanguinarine concentration of 0.339%, which is over 200 times the sanguinarine concentration of adulterated massage oil (submitted and under peer review). Although black salve is usually applied to an isolated skin lesion, some patients have simultaneously applied black salve to multiple sites, thus increasing the surface area for sanguinarine exposure (Krishnamurthy K, 2009). In addition, black salve usually has a prolonged 24 hour contact time with the skin. As epidemic dropsy can occur from transcutaneous adulterated mustard oil application, it is reasonable to suggest that black salve, with its greater sanguinarine concentration and greater contact time, may have the potential to induce epidemic dropsy.

Clinical presentation and pathophysiology of epidemic dropsy An outbreak of epidemic dropsy is usually heralded by the onset of leg oedema in a previously unaffected community that use mustard oil for cooking (Sachdev et al., 1988). Epidemic dropsy effects a number of body systems and can have a diverse clinical presentation (Figure 2). The core pathogenic mechanism underlying the disease occurs at the capillary level where a neovascular process appears to develop gradually due to argemone oil exposure. New vessel formation with endothelial cell proliferation around dilated capillaries occurs (Lal et al., 1941). These histological findings have been found in lung, kidney, liver, heart and skin tissue from rats, monkeys and humans exposed to argemone oil (Bhende, 1956; Chakravarty et al., 1955; Verma et al., 2001).

In experiments where rabbits were exposed to argemone alkaloids, there was evidence of growth of a fragile vessel plexus prone to haemorrhage, with small vessel dilatation and increased capillary permeability (Chakravarty et al., 1955; Chauduri, 1959). However, the processes leading to these pathological changes have not been definitively elucidated. Neovascularization is also a feature of other human diseases, most notably diabetes. Gradual capillary damage by elevated glucose levels results in the formation of diabetic retinopathy in 40% of type II diabetes mellitus patients six years after diagnosis (Lee et al., 2015). The clinical features of epidemic dropsy begin approximately 20 days after contaminated oil ingestion (Tandon et al., 1975), with different systems affected at various time points in the course of the illness. The gradual toxicity of argemone oil may be explained by the time required for cumulative capillary damage to occur, although this process appears more rapid than that which occurs in diabetic patients.

Epidemic dropsy usually has an insidious onset with watery diarrhoea, anorexia, low grade fever, nausea and vomiting lasting from a few days to more than a week being reported in most cases (Sinani, 1976). This usually preceeds the onset of peripheral oedema, which may

Bilateral symmetrical pitting oedema of the lower limbs, that can extend from the ankles up to the pelvis and abdominal wall, is a dominant feature of the condition (Singh et al., 2000). The oedema worsens with standing and is associated with itching, burning and paraesthesia. The oedema is relatively resistant to diuretics and may become more firm with time. In 25% of patients the oedema lasts longer than two months, while for 10% it lasts beyond five months (Wadia et al., 1971). A tender, erythematous rash with bluish mottling (erythrocyanosis) occurs over oedematous areas. Altered skin pigmentation in these areas can also occur. Telangiectasias and nodular sarcoid like lesions may develop on the skin and internal mucous membranes (Kar et al., 2001). These can bleed, sometimes excessively. Most patients with epidemic dropsy develop a normocytic normochromic anaemia that has a multifactorial origin due to bleeding from the gastrointestinal tract, bone marrow suppression and shortened red cell life span (Sengupta & Napier, 1940).

Cough and breathlessness are common symptoms. Exudation of protein-rich fluid from pulmonary capillaries into interstitial alveolar tissues produces pulmonary oedema of non-cardiac origin with manifestations of hypoxia, respiratory alkalosis, increased alveolar-arterial gradient and deranged diffusion capacity (Rangan & Barceloux, 2009). In severe cases pulmonary hypertension develops leading to elevated right ventricular systolic pressure, right-sided cardiac chamber dilatation, right sided heart failure, and congestive hepatomegaly (Chopra & Basu, 1930).

High output cardiac failure with a wide pulse pressure, tachycardia, orthopnoea and gallop rhythm can result from the peripheral vasodilatory effects of AO. An apical systolic murmur due to functional mitral incompetence may occur (Sanghvi et al., 1960), with flow murmurs being common due to associated anaemia and a high output state.

Elevated glomerular permeability can result in hypoalbuminaemia and proteinurea, fluid extravasation resulting in hypovolaemia with renal insufficiency that can develop into acute renal failure (Prakash, 1999). Increased organ vascularity and congestion with transudate collections in the pleural, pericardial and peritoneal spaces, due to increased capillary permeability, are features usually found at autopsy (Verma et al., 2001).

Ophthalmology features occur relatively late in the course of epidemic dropsy with some studies reporting 11% of patients developing glaucoma accompanied by a raised intraocular pressure (IOP) >22mmHg (Sachdev et al., 1988). Patients also experience visual field defects where the extent of visual field loss correlates more with the severity of the systemic disease

This article is protected by copyright. All rights reserved. than the level of IOP elevation. This suggests that the pathogenic process may be more related to retinal damage from an argemone oil-induced leaking microangiopathy than a direct effect of elevated pressures (Singh et al., 2006). Other visual complications include subconjunctival haemorrhage, superficial retinal haemorrhages, retinal venous dilatation and tortuosity, subhyaloid haemorrhages, macular and papillary oedema and central retinal vein occlusion. Visual field defects are rarely permanent and intraocular pressures usually return to normal values within 12 weeks (Malik et al., 2004).

The mortality rate for epidemic dropsy lies between 5-10% (Park, 2007; Sainini & Abraham, 1999). Death primarily results from cardiac arrest secondary to congestive cardiac failure, noncardiogenic pulmonary oedema, pericardial fluid accumulation or renal failure (Chopra & Basu, 1930). However, the majority of patients completely recover by 3 months.

Patients that develop epidemic dropsy from transcutaneous argemone oil exposure have had mild cases of the condition. They developed bilateral pedal oedema of 3-7 days duration with erythema, rash and tenderness. Two patients with higher sanguinarine serum levels (3.5 and 4µg/100ml) developed gastro-intestinal symptoms of nausea, anorexia and diarrhoea in addition to non-tender hepatomegaly and dilated, tortuous retinal veins. The patient with the highest serum concentration developed a peripapillary superficial retinal haemorrhage. Although three patients experienced a widened pulse pressure no cardiovascular or respiratory symptoms were present. A normocytic, hypochromic anaemia was found in all cases whilst kidney and liver function tests were normal. These clinical findings confirm earlier evidence in monkeys that argemone oil is absorbed through intact healthy skin and can induce the topical and internal organ changes of epidemic dropsy (Chakravarty & Chaudhuri, 1951).

Does sanguinarine cause epidemic dropsy? The potential exists for topical agents that contain sanguinarine to be systemically absorbed, resulting in acute toxicity and illness. Although toxins are more poorly absorbed through the skin, the heating of AO associated with cooking reduces much of its toxicity (Chakravarty et al., 1955), this may explain how similar plasma sanguinarine concentrations can be generated either with ingestion or transcutaneous exposure. In order to determine whether black salve may cause epidemic dropsy, it is necessary to establish the role of sanguinarine in the pathogenesis of the disease.

Systemic oxidative stress plays an important role in epidemic dropsy. A free radical is a molecule that can exist independently with one or more unpaired electrons (Halliwell & Gutteridge, 2007). In living systems, free radicals are predominantly represented as reactive

This article is protected by copyright. All rights reserved. - oxygen species (ROS). The most common ROS being: superoxide anion radical (O2˙ ), 1 peroxide, hydroxyl radical (˙OH) and singlet oxygen ( ΔgO2) an excited state of molecular oxygen (Halliwell, 1991).

A significant source of endogenous ROS comes from the byproduct of oxidative metabolism in the mitochondria where adenosine triphosphate (ATP) is generated from glucose (Cadenas & Davies, 2000). Electrons pass through 4 complexes of the electron transport chain to - generate ATP and water. As a side reaction molecular oxygen is converted to O2˙ , a potent - ROS (Turrens, 1997). O2˙ is converted into hydrogen peroxide H2O2 (a ROS that is not a free

radical) by spontaneous conversion or superoxide dismutase. H2O2 is the key agent in the Fenton reaction, which readily occurs in the presence of metal catalysts (iron or copper) and produces ˙OH, one of the most unstable ROS that exists in biological systems (Buettner, 1993). The half-life of ˙OH is so short (10-9 seconds) that it exerts its damaging effects almost exclusively at the site of its generation (Sies, 1993).

ROS production can be induced by the action of environmental agents such as the sanguinarine contained in AO (Das et al., 2005). Sanguinarine has been shown to generate ROS in vitro by rapidly cycling between its oxidized and reduced form intracellularly and on - cell membranes, resulting in significant H2O2 and to a lesser extent O2˙ generation (Matkar et al., 2008). This appears to occur via sanguinarine adduct formation with nicotinamide adenine dinucleotide (reduced form, NADH), this adduct subsequently transforming to DHSG with the generation of ROS. DHSG is then spontaneously re-oxidized to sanguinarine, forming a metabolic loop that significantly diminishes the pool of intracellular NADH and nicotinamide adenine dinucleotide phosphate (reduced form, NADPH) (Sandor et al., 2018). The ROS induced by sanguinarine (Das et al., 1991; Maiti & Chatterjee, 1995), add carbonyl residues to cell membrane proteins (Amici et al., 1989) impairing membrane transport and altering permeability (Davies, 1993; Hebbel, 1986), a characteristic of epidemic dropsy.

The human body contains a number of bio-antioxidants that give protection against ROS such as vitamin C, vitamin E, reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and gamma glutamyl transpeptidase (GGT) (Kehrer, 1993). Significant oxidative stress depletes anti-oxidants enabling oxidative damage to lipids, DNA and proteins.

Lipid peroxidation is frequently used to assay the extent of oxygen free radical damage. Patients with epidemic dropsy have lipid peroxide levels elevated by 175% with superoxide - dismutase and catalase, the respective scavengers of O2˙ and H2O2, being decreased 37 and 61% (Das et al., 2005). This anti-oxidant depletion correlates with the ROS profile generated by cellular sanguinarine exposure (Matkar et al., 2008). Non-enzymatic antioxidants are also

This article is protected by copyright. All rights reserved. reduced with vitamin E, retinol and glutathione being significantly decreased by 46, 61 and 57%, and the entire antioxidant pool in epidemic dropsy patients being reduced by 53% (Das et al., 2005). This, in addition to the amelioration of epidemic dropsy symptoms in patients administered antioxidants (Kumar et al., 1992), suggests oxidative stress plays a dominant role in epidemic dropsy pathogenesis.

In addition to experimental evidence showing sanguinarine induces ROS, the alkaloid has a number of other effects likely to contribute to the pathophysiology of epidemic dropsy. Sanguinarine blocks pyruvic acid metabolism, elevating blood pyruvate levels (Kirwan, 1934). Pyruvate is a known vasodilator and endothelial toxin that increases capillary permeability, likely contributing to interstitial fluid accumulation.

Sanguinarine inhibits the intestinal Na+/K+-ATPase pump, impairing the active transport of glucose across the intestinal barrier (Tandon et al., 1993), likely contributing to the gastrointestinal symptoms of epidemic dropsy. Sanguinarine also inhibits the Na+/K+-ATPase pump in guinea pig cardiac myocytes, increasing intracellular Ca2+ levels with a 10µM dose having a positive inotropic effect (Seifen et al., 1979). Pharmacokinetic studies of sanguinarine in rats have suggested that oral sanguinarine bioavailability is low with 42% of 3H-sanguinarine remaining in the gastrointestinal tract and a low sanguinarine to DHSG plasma concentration ratio suggesting most sanguinarine is metabolized to DHSG during or

soon after its absorption from the gastrointestinal tract with both compounds having a Tmax of 2 hours (Vecera et al., 2007). As DHSG does not appear to interact with the Na+/K+-ATPase pump, it has been argued that oral sanguinarine, due to its poor absorption and rapid metabolism to DHSG, should have a limited ability for in vivo Na+/K+-ATPase inhibition (Janovska et al., 2010). Epidemic dropsy patients have significantly elevated hydroxybutyrate dehydrogenase (HBDH) levels (an enzyme found in myocardium) of 165 U/L compared to controls 97 U/L, this remaining elevated one month after argemone oil exposure cessation at 128U/L. Sanguinarine due to its ability to act on cardiac glycoside receptors has been suggested as a possible cause of the myocardial toxicity seen in these patients (Gohil et al., 2011; Vecera et al., 2007).

The iminium form of sanguinarine also inhibits the Na+/K+-ATPase pump of red blood cells, increasing their osmotic fragility, while the alkanolamine form produces red cell lysis without altering electrolyte transport (Cala et al., 1982). Normocytic anaemia is a constant feature of epidemic dropsy (Narayan et al., 2000) and may contribute to the breathlessness found in AO intoxication.

This article is protected by copyright. All rights reserved. Ten epidemic dropsy patients with sanguinarine serum levels ranging from 0.0136 to 0.782 µg/ml showed a significant (p<0.001) positive (r=0.80) correlation between sanguinarine concentration and serum malondialdehyde (MDA) levels suggesting a link between sanguinarine and the induction of oxidative stress (Banerjee et al., 2000). The authors also reported the severity of disease seemed dependent on sanguinarine levels. Another study investigated 22 patients with epidemic dropsy, the two with measurable blood sanguinarine levels being most severely affected (Shenolikar et al., 1974). Sanguinarine may however be an indicator of the level of AO exposure rather than the causative toxic agent. There is a significant difference between the toxicity of AO and equivalent concentrations of

sanguinarine in animal studies. The oral LD50 for AO in rats corresponds to a 5mg/kg sanguinarine body weight dose, while the oral LD50 of pure sanguinarine independent of AO is 1658 mg/kg body weight (Becci et al., 1987). In rats an oral dose of AO containing only 0.55mg sanguinarine/kg body weight causes toxicity (Babu et al., 2006). Sangrovit® is a livestock weight gain feed additive that contains sanguinarine but not other constituents derived from Argemone mexicana. Pigs fed Sangrovit® with a sanguinarine concentration of 1.28mg/ kg feed for 90 days had sanguinarine plasma levels as high as 0.11 µg/ml, comparable to the toxic range of 0.01-0.78 µg/ml reported in patients with epidemic dropsy (Banerjee et al., 2000), and remained healthy with no manifestations of toxicity (Kosina et al., 2004). This suggests the existence of other factors present in AO that may act either as toxins themselves or potentiate the toxicity of sanguinarine.

Total alkaloids from argemone seeds have been injected into the ears of rabbits at an increasing dose of 2-4mg/kg to 15mg/kg over six to fourteen weeks. Compared to control animals, the treated animals developed intercommunicating dilated vessel plexuses that formed sinusoids that had a marked tendency to haemorrhage (Chakravarty et al., 1955), such neovascularization is a key pathological feature found in epidemic dropsy cases. The method for alkaloid isolation however was not described preventing a determination as to whether non alkaloid constituents were present in the injections. The acute dermal toxicity of sanguinarine has been studied in rabbits, where a dose of 200mg/kg was found to be

non-lethal, this prevented the determination of a sanguinarine dermal LD50 value. Anorexia and diarrhoea occurred in 40% of animals, with gross necropsy revealing no evidence of organ effects. The failure of large topical sanguinarine doses to induce an epidemic dropsy type condition in rabbits suggests sanguinarine in isolation as a topical therapy constituent would be unlikely to produce epidemic dropsy in humans (Becci et al., 1987).

Studies in monkeys have similarly shown the induction of an epidemic dropsy type toxicity following oral, intramuscular and topical argemone oil exposure. Oral administration of low 0.5% argemone oil concentrations over a six month period showed a cumulative toxic effect

This article is protected by copyright. All rights reserved. (Chaudhubi & Chakbavauty, 1953). Epidemic dropsy features did not however occur when monkeys were administered oral sanguinarine at 1.6mg/kg of body weight (Shenolikar et al., 1974). A male monkey that had 1ml of argemone oil administered to shaved abdominal skin 25 times (total 25mls) over the course of a month died after developing an epidemic dropsy type toxicity syndrome, with this finding confirmed by topical argemone oil testing on several other monkeys. This result further strengthens the hypothesis that argemone oil is absorbed through intact skin and can result in epidemic dropsy. The authors also noted that a human sweeper involved in holding the monkeys had accidentally been exposed to a few brushes of argemone oil on the backs of his hands. He developed skin erythema that blanched on pressure similar to the early cutaneous manifestations of epidemic dropsy (Chakravarty & Chaudhuri, 1951; Shenolikar et al., 1974)

Human argemone oil toxicity studies were conducted in the early 20th century. Individuals fed food cooked with 2-10% argemone oil adulterated mustard oil developed epidemic dropsy after a period ranging from 6 to 21 days of exposure, while those cooked food with uncontaminated mustard oil did not (Chopra et al., 1939).

What argemone oil compound/s are responsible for epidemic dropsy toxicity? The focus of AO toxicity studies has been centred on its alkaloid constituents, a chemical class with a history of human physiological effects. DHSG constitutes 87% of the alkaloids in AO, with sanguinarine contributing 5% to the alkaloid pool. AO toxicity has been attributed to both sanguinarine and DHSG with one review reporting the toxicity of sanguinarine being two and a half times greater than the toxicity of DHSG (Das & Khanna, 1997). This paper however, appears to have incorrectly referenced earlier work by Sarkar et al published in Nature in 1948. The original paper stated “When sanguinarine hydrochloride was injected intraperitoneally to albino rats in a dose of 2 mgm/ 100 gm body-weight, there was 100 per cent mortality. In contrast to this, dihydrosanguinarine at about two and a half times this dose, or oxysanguinarine at about four times this dose, did not kill any of the rats injected” (Sarkar, 1948). The suggestion that DHSG is a toxic, albeit less potent compound than sanguinarine based on the work of Sarkar et al is incorrect.

In rats, DHSG toxicity has been assessed (Vrublova et al., 2008). Oral DHSG was administered at a concentration of 500 ppm (highest concentration group with an average of 58mg/per kg rat body weight/day) for 3 months, resulting in plasma DHSG levels up to 0.71 ng/ml. No abnormalities in biochemistry or haematology parameters were found, nor was detectable oxidative stress induced. As the morphology of several tissues examined histologically correlated with that of control animals, the authors concluded there was no

This article is protected by copyright. All rights reserved. evidence of DHSG induced systemic toxicity with 0.5% DHSG oral exposure. This however does not exclude DHSG as a causative compound in epidemic dropsy. Mustard oil with AO contamination between 1.22-8.77%, the majority of which would be DHSG, has been found in households that have developed epidemic dropsy (Ghosh et al., 2005). Depending on how much oil is used in cooking and remains in the food, significantly higher oral DHSG exposures that were not assessed in this study could result. The maximum rat DHSG plasma concentration of 0.71ng/ml reached in this study is approximately 1000 times lower than the plasma sanguinarine concentrations reported in patients with epidemic dropsy (Banerjee et al., 2000) further suggesting the dose was inadequate for the purpose of excluding a pathogenic role for DHSG in epidemic dropsy. While DHSG exposed animals were found to have tissue histology that correlated with control animals, it is interesting to note that tiny foci of oedema in ileal vili submucosal connective tissues and minor foci of hyperaemia and capillary dilatation in the kidneys were reported in animals exposed to 500ppm oral DHSG. These minor histological changes reflect the type of vascular pathology observed in epidemic dropsy cases. Whether exposure to higher concentrations of DHSG would result in more pathognomonic changes is currently unknown.

Apart from the alkaloid compounds present in argemone oil, other constituents may contribute to the pathogenesis of epidemic dropsy. In 1971 a solid lipid fracton was extracted from AO and described as a sanguinarine potentiator (Rukmini, 1971). Individually, both sanguinarine, and AO from which the sanguinarine had been removed, showed no toxicity for rhesus monkeys. When combined, the monkeys developed symptoms of epidemic dropsy (Rukmini, 1971). The fatty acid composition of this lipid fraction was given various identifications of: (+) 6-hydroxy-6-methyl- 9-oxo-octacosanoic acid (Rukmini, 1975), an 11-hydroxy-triacontanoic acid and 11-oxo-octacosanoic acid (90:10) mix (Mahato et al., 1975), and a 9- and 11-oxo-octacosanoic and 11-oxotriacontanoic acid (1:2:1) (Figure 1) mix (Gunstone et al., 1977). In 1993 the fatty acid composition of this lipid fraction of A. mexicana was determined using GC-MS as 11-oxo-octacosanoic 45% and methyl 11-oxo-triacontanoic acid 25%, with lower concentrations of other fatty acids (Fletcher et al., 1993). Although the mechanism of argemone oil toxicity is still not clear, these unsual fatty acids likely play a significant role in epidemic dropsy pathogenesis and warrant careful study.

Conclusion Patients can develop epidemic dropsy from the topical application of sanguinarine containing adulterated mustard oil. While black salve contains significantly greater sanguinarine concentrations and is in contact with the skin for longer periods of time, it appears the

This article is protected by copyright. All rights reserved. development of epidemic dropsy is dependent on the presence of both sanguinarine and other AO constituents. The theoretical risk of patients developing epidemic dropsy from the use of black salve is likely to be low. No cases of epidemic dropsy have ever been reported from black salve use. Further research is required to assess the topical pharmacokinetics of sanguinarine, DHSG and other argemone oil and black salve constituents. Studies also should be conducted to determine the pathophysiological mechanisms responsible for epidemic dropsy, including higher concentration DHSG and fatty acid constituent toxicity studies. Although epidemic dropsy is a condition known to the medical profession for over 100 years and continues to effect communities, it remains a condition incompletely understood.

This article is protected by copyright. All rights reserved. References Amici, A., Levine, R. L., Tsai, L., & Stadtman, E. R. (1989). Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions. Journal of Biological Chemistry, 264(6), 3341.

Babu, C. K., Khanna, S. K., & Das, M. (2006). Safety evaluation studies on argemone oil through dietary exposure for 90 days in rats. Food and Chemical Toxicology, 44(7), 1151-1157. doi:10.1016/j.fct.2006.02.003

Banerjee, B. D., Seth, V., Koner, B. C., Ahmed, R. S., Sharma, M., Grover, S. S., . . . Pasha, S. T. (2000). Evaluation of oxidative stress in some cases of argimone oil poisoning during a recent outbreak of epidemic dropsy in India. International Journal of Environmental Health Research, 10(4), 341-346.

Becci, P. J., Schwartz, H., Barnes, H. H., & Southard, G. L. (1987). Short-term toxicity studies of sanguinarine and of two alkaloid extracts of Sanguinaria canadensis L. J Toxicol Environ Health, 20(1-2), 199-208. doi:10.1080/15287398709530972

Bhende, Y. M. (1956). Vascular changes prodcued by argemone oil. 2. Changes in rats given injections of the oil. Journal of Postgraduate Medicine, 2, 185-193.

Brahmachari, G., Gorai, D., & Roy, R. (2013). Argemone mexicana: chemical and pharmacological aspects. Revista Brasileira de Farmacognosia, 23(3), 559-567.

Buettner, G. R. (1993). The Pecking Order of Free Radicals and Antioxidants: Lipid Peroxidation, α-Tocopherol, and Ascorbate. Archives of Biochemistry and Biophysics, 300(2), 535-543. doi:10.1006/abbi.1993.1074

Cadenas, E., & Davies, K. J. A. (2000). Mitochondrial free radical generation, oxidative stress, and aging 1 1 This article is dedicated to the memory of our dear friend, colleague, and mentor Lars Ernster (1920–1998), in gratitude for all he gave to us. Free Radical Biology and Medicine, 29(3), 222-230. doi:10.1016/S0891-5849(00)00317-8

Cala, P. M., Norby, J. G., & Tosteson, D. C. (1982). Effects of the plant alkaloid sanguinarine on cation transport by human red blood cells and lipid bilayer membranes. J Membr Biol, 64(1-2), 23-31.

Chakravarty, N. K., & Chaudhuri, R. N. (1951). Production of epidemic dropsy in monkeys. The Indian Medical Gazette, 86, 392-395.

This article is protected by copyright. All rights reserved. Chakravarty, N. K., Chaudhuri, R. N., & Werner, G. (1955). Observations on vascular changes produced by argemone alkaloids. The Indian journal of medical research, 43(1), 107.

Chaudhubi, E. N., & Chakbavauty, N. K. (1953). Vascular pattern in Agremone fed animals. Indian Journal of Medical Sciences, 7(7), 334-337.

Chauduri, R. (1959). Clinical and experimental research. Bull. Cal. Sch. Trop. Med, 7, 157-161.

Chopra, R. N., & Basu, U. P. (1930). Cardiovascular Manifestations of Epidemic Dropsy and their Treatment. Indian Medical Gazette, 65(10), 546-550.

Chopra, R. N., Pasricha, C. L., Goyal, R. K., Lal, S., & Sen, A. K. (1939). The Experimental Production of Syndrome of Epidemic Dropsy in Man. The Indian Medical Gazette, 74(4), 193-195.

Croaker, A., King, G. J., Pyne, J. H., Anoopkumar-Dukie, S., & Liu, L. (2016). Sanguinaria canadensis: Traditional Medicine, Phytochemical Composition, Biological Activities and Current Uses. International Journal of Molecular Sciences, 17(9), 1414.

Croaker, A., King, G. J., Pyne, J. H., Anoopkumar-Dukie, S., & Liu, L. (2017a). A Review of Black Salve: Cancer Specificity, Cure, and Cosmesis. Evidence-Based Complementary and Alternative Medicine, 2017, 11. doi:10.1155/2017/9184034

Croaker, A., King, G. J., Pyne, J. H., Anoopkumar-Dukiec, S., Simanek, V., & Liu, L. (2017b). Carcinogenic potential of sanguinarine, a phytochemical used in ‘therapeutic’ black salve and mouthwash. Mutation Research-Reviews in Mutation Research, 774, 46-56.

Das, M., Babu, K., Reddy, N. P., & Srivastava, L. M. (2005). Oxidative damage of plasma proteins and lipids in epidemic dropsy patients: Alterations in antioxidant status. Biochimica Et Biophysica Acta-General Subjects, 1722(2), 209-217. doi:10.1016/j.bbagen.2004.12.014

Das, M., & Khanna, S. (1998). Epidemic dropsy. The National medical journal of India, 11(5), 207.

Das, M., & Khanna, S. K. (1997). Clinicoepidemiological, toxicological, and safety evaluation studies on argemone oil. Critical Reviews in Toxicology, 27(3), 273-297. doi:10.3109/10408449709089896

This article is protected by copyright. All rights reserved. Das, M., Upreti, K. K., & Khanna, S. K. (1991). Biochemical toxicology of argemone oil. Role of reactive oxygen species in iron catalyzed lipid peroxidation. Bulletin of environmental contamination and toxicology, 46(3), 422-430. doi:10.1007/BF01688942

Davies, K. J. (1993). Protein modification by oxidants and the role of proteolytic enzymes. Biochemical Society transactions, 21(2), 346.

Fletcher, M. T., Takken, G., Blaney, B. J., & Alberts, V. (1993). Isoquinoline Alkaloids and Keto-Fatty Acids of Argemone-Ochroleuca and A-Mexicana (Mexican Poppy) Seed. 1. An Assay-Method and Factors Affecting their Concentration. Australian Journal of Agricultural Research, 44(2), 265-275. doi:10.1071/ar9930265

Ghosh, P., Reddy, M. M. K., & Sashidhar, R. B. (2005). Quantitative evaluation of sanguinarine as an index of argemone oil adulteration in edible mustard oil by high performance thin layer chromatography. Food Chemistry, 91(4), 757-764. doi:10.1016/j.foochem.2004.10.012

Gohil, J. R., Mankad, B. D., & Amin, B. K. (2011). Sanguinarine, hydroxybutyrate dehydrogenase and fatigue in epidemic dropsy: A retrospective study of an outbreak and its control from gujarat, India. WebmedCentral MEDICINE, 2(12), WMC002118.

Gomber, S., Daral, T., Sharma, P., & Faridi, M. (1994). Epidemic dropsy in Trans Yamuna areas of Delhi and UP. Indian pediatrics, 31, 671-671.

Gunstone, F. D., Holliday, J. A., & Scrimgeour, C. M. (1977). Fatty acids, part 51. The long-chain oxo acids (argemonic acid) in Argemone mexicana seed oil. Chemistry and Physics of Lipids, 20(4), 331-335.

Halliwell, B. (1991). Reactive oxygen species in living systems: source, biochemistry, and role in human disease. The American journal of medicine, 91(3), S14-S22.

Halliwell, B., & Gutteridge, J. M. C. (2007). Free radicals in biology and medicine (Vol. 4th). Oxford;New York;: Oxford University Press.

Hebbel, R. P. (1986). Erythrocyte antioxidants and membrane vulnerability. The Journal of laboratory and clinical medicine, 107(5), 401.

Janovska, M., Kubala, M., Simanek, V., & Ulrichova, J. (2010). Interaction of sanguinarine with the Na+/K+-ATPase. Febs Journal, 277, 207-208.

This article is protected by copyright. All rights reserved. Kar, H., Jain, R., Sharma, P., Gautam, R., Kumar, P., & Bhardwaj, M. (2001). Epidemic dropsy: A study of cutaneous manifestations with histopathological correlation. Indian Journal of Dermatology, Venereology, and Leprology, 67(4), 178.

Kehrer, J. P. (1993). Free Radicals as Mediators of Tissue Injury and Disease. CRC Critical Reviews in Toxicology, 23(1), 21-48. doi:10.3109/10408449309104073

Kirwan, E. W. O. G. (1934). Primary glaucoma: A symptom complex of epidemic dropsy. Archives of Ophthalmology, 12(1), 1-20. doi:10.1001/archopht.1934.00830140009001

Kosina, P., Walterová, D., Ulrichová, J., Lichnovský, V., Stiborová, M., Rýdlová, H., . . . Šimánek, V. m. (2004). Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding experiment. Food and Chemical Toxicology, 42(1), 85-91.

Krishnamurthy K, H. C., Del Priore J. (2009). Bloodroot necrosis. Journal of the American Academy of Dermatology, 60(3), AB44-AB44. doi:10.1016/j.jaad.2008.11.213

Kumar, A., Husain, F., Das, M., & Khanna, S. K. (1992). An out-break of epidemic dropsy in the Barabanki District of Uttar Pradesh, India: a limited trial for the scope of antioxidants in the management of symptoms. Biomed Environ Sci, 5(3), 251-256.

Lal, R. B., Chatterjee, S. R., Agarwal, S. P., & Das Gupta, A. C. (1941). Investigation into epidemiology of epidemic dropsy: biological test of specific toxin in sample of oil. Indian Journal of Medical Research, 29, 167.

Lal, R. B., & Dasgupta, R. B. (1942). Investigation into the epidemiology of Epidemic dropsy: incidence by season. Indian Journal of Medical Research, 30, 145-150.

Lal, R. B., & Roy, S. C. (1937). Investigations into the epidemiology of epidemic dropsy. Indian Journal of medical researhc, 25, 163-176.

Lee, R., Wong, T. Y., & Sabanayagam, C. (2015). Epidemiology of diabetic retinopathy, diabetic macular edema and related vision loss. Eye and Vision, 2, 17. doi:10.1186/s40662-015-0026-2

Mahato, S., Sahu, N., Narayanaswami, A., Chakravarti, R., & Chakravarti, D. (1975). Structures of fatty acids from the oil of Argemone mexicana. Journal.

Maiti, M., & Chatterjee, A. (1995). Production of singlet oxygen by sanguinarine and berberine. Current Science, 68(7), 734-736.

This article is protected by copyright. All rights reserved. Malik, K., Dadeya, S., Gupta, V., Sharan, P., & Dhawan, M. (2004). Pattern of intraocular pressure in epidemic dropsy in India. Tropical doctor, 34(3), 161-162.

Manson-Bahr, P., & Apted, F. (1982). Plant poisons. In Manson's Tropical Diseases (pp. 571-572): The English Language Book Society and Baillière Tindall London.

Matkar, S. S., Wrischnik, L. A., & Hellmann-Blumberg, U. (2008). Production of hydrogen peroxide and redox cycling can explain how sanguinarine and chelerythrine induce rapid apoptosis. Archives of Biochemistry and Biophysics, 477(1), 43-52. doi:10.1016/j.abb.2008.05.019

Narasimhan, C., T, J. G., Thomas, G., Israel, J., Rao, P. S., & Pulimood, B. M. (1991). Epidemic dropsy in Andhra Pradesh due to contaminated ghee. J Assoc Physicians India, 39(10), 749-750.

Narayan, S., Sharma, S., & Kaur, M. (2000). Evaluation of hematological parameters in epidemic dropsy. Indian Journal of Hematology and Blood Transfusion, 18(1), 8-10.

Park, K. (2007). Park's textbook of preventive and social medicine.

Prakash, J. (1999). Acute renal failure in epidemic dropsy. Renal failure, 21(6), 707-711.

Rangan, C., & Barceloux, D. G. (2009). Food contamination. Dis Mon, 55(5), 263-291. doi:10.1016/j.disamonth.2009.01.003

Rukmini, C. (1971). Sanguinarine Potentiating Factor in Argemone Oil. Indian Journal of Medical Research, 59(10), 1676-&.

Rukmini, C. (1975). New, unusual long chain fatty acid (argemonic acid) from Argemone Mexicana. Journal of the American Oil Chemists' Society, 52(6), 171.

Sachdev, M. S., Sood, N. N., Verma, L. K., Gupta, S. K., & Jaffery, N. F. (1988). Pathogenesis of epidemic dropsy glaucoma. Arch Ophthalmol, 106(9), 1221-1223.

Sainini, G. S., & Abraham, P. (1999). API text book of medicine: Association of Physicians of India.

Sandor, R., Slanina, J., Midlik, A., Sebrlova, K., Novotna, L., Carnecka, M., . . . Pes, O. (2018). Sanguinarine is reduced by NADH through a covalent adduct. Phytochemistry, 145, 77-84. doi:10.1016/j.phytochem.2017.10.010

This article is protected by copyright. All rights reserved. Sanghvi, L., Misra, S., & Bose, T. (1960). Cardiovascular manifestations in Argemone mexicana poisoning (epidemic dropsy). Circulation, 21(6), 1096-1106.

Santavy, F. (1970). The Alkaloids. Chemistry and Physiology. New York, London, 333-454.

Sanyal, P. (1950). Argemone and mustard seeds. Indian medical gazette, 85(11), 498-500.

Sarkar, S. M. (1948). Isolation from argemone oil of dihydrosanguinarine and sanguinarine; toxicity of sanguinarine. Nature, 162(4111), 265.

Schneider, J., Azazh, A., Bane, A., & Seboxa, T. (2013). Epidemic dropsy: case report and the morphologic features in a patient who died at Tikur Anbessa Specialized Hospital. Ethiop Med J, Suppl 2, 33-37.

Seifen, E., Adams, R. J., & Riemer, R. K. (1979). Sanguinarine: a positive inotropic alkaloid which inhibits cardiac Na+,K+-ATPase. Eur J Pharmacol, 60(4), 373-377.

Sengupta, P. C., & Napier, L. E. (1940). Haematological Changes in Epidemic Dropsy. Indian Journal of Medical Research, 28(1), 197-206.

Sharma, B., Malhotra, S., Bhatia, V., & Rathee, M. (1999). Epidemic dropsy in India. Postgraduate medical journal, 75(889), 657-661.

Shenolikar, I. S., Rukmini, C., Krishnamachari, K. A., & Satyanarayana, K. (1974). Sanguinarine in the blood and urine of cases of epidemic dropsy. Food Cosmet Toxicol, 12(5-6), 699-702.

Sies, H. (1993). Strategies of antioxidant defense. European Journal of Biochemistry, 215, 213-219. doi:10.1016/1043-6618(95)86904-2

Simanek, V. (1985). Benzophenanthridine alkaloids. The alkaloids, 26, 185-240.

Sinani, G. S. (1976). Epidemic Dropsy in Progress in clinical medicine in India. 92-104.

Singh, K., Jot Singh, M., & Chander Das, J. (2006). Visual field defects in epidemic dropsy. Clinical Toxicology, 44(2), 159-163.

Singh, N., Anuradha, S., Dhanwal, D., Singh, K., Prakash, A., Madan, K., & Agarwal, S. (2000). Epidemic dropsy--a clinical study of the Delhi outbreak. The Journal of the Association of Physicians of India, 48(9), 877-880.

Sood, N. N., Sachdev, M. S., Mohan, M., Gupta, S. K., & Sachdev, H. P. (1985). Epidemic dropsy following transcutaneous absorption of Argemone mexicana oil. Trans R Soc Trop Med Hyg, 79(4), 510-512.

Steyn, D. (1950). Poisoning with the Seeds of Argemone mexicana (Mexican Poppy) in Human Beings. Indian Epidemic Dropsy in South Africa. South African Medical Journal, 24(18), 333-339.

Tandon, R. K., Singh, D. S., Arora, R. R., Lal, P., & Tandon, B. N. (1975). Epidemic dropsy in New Delhi. Am J Clin Nutr, 28(8), 883-887.

Tandon, S., Das, M., & Khanna, S. K. (1993). Effect of sanguinarine on the transport of essential nutrients in an everted gut sac model: role of Na+,K(+)-ATPase. Natural toxins, 1(4), 235-240. doi:10.1002/nt.2620010406

Thatte, U., & Dahanukar, S. (1999). The Mexican poppy poisons the Indian mustard facts and figures. J Assoc Physicians India, 47(3), 332-335.

Turrens, J. F. (1997). Superoxide Production by the Mitochondrial Respiratory Chain. Bioscience Reports, 17(1), 3-8. doi:10.1023/A:1027374931887

Upreti, K. K., Das, M., & Khanna, S. K. (1991). Biochemical toxicology of argemone oil. I. effect on hepatic cytochrome P‐450 and xenobiotic metabolizing enzymes. Journal of Applied Toxicology, 11(3), 203-209.

Vecera, R., Klejdus, B., Kosina, P., Orolin, J., Stiborova, M., Smrcek, S., . . . Simanek, V. (2007). Disposition of sanguinarine in the rat. Xenobiotica, 37(5), 549-558. doi:10.1080/00498250701230542

Verma, S., Dev, G., Tyagi, A., Goomber, S., & Jain, G. (2001). Argemone mexicana poisoning: autopsy findings of two cases. Forensic science international, 115(1), 135-141.

Vrublova, E., Vostalova, J., Vecera, R., Klejdus, B., Stejskaj, D., Kosina, P., . . . Ulrichova, J. (2008). The toxicity and pharmacokinetics of dihydrosanguinarine in rat: A pilot study. Food and Chemical Toxicology, 46(7), 2546-2553. doi:10.1016/j.fct.2008.04.013

This article is protected by copyright. All rights reserved. Wadia, R., Relwani, G., Batra, R., Ichaporia, R., & Grant, K. (1971). Epidemic dropsy in Poona 1969 (clinical features and 1 year follow up). The Journal of the Association of Physicians of India, 19(9), 641.

Figure Legends

Figure 1. Chemical structures of major alkaloids and fatty acids in argemone oil

Figure 2. Clinical presentation of epidemic dropsy