The Paradox of Human Equivalent Dose Formula: A Canonical Case Study of Abrus Precatorius Aqueous Leaf Extract in Monogastric Animals

Abstract There is abundant literature on the toxicity of A. precatorius seeds. However there is a need to define the toxicity limit of the Abrus precatorius leaf in monogastric animals. Human Equivalent Dose (HED) which is equal to animal dose multiplied by animal km (metabolism constant) divided by human km was used to project the LD50 of fifteen monogastric animals, where human km factor is body weight (kg) divided by body surface area (m2). Human Equivalent No-observable Adverse Effect Doses were determined by multiplying the animal no-observable adverse effect dose by animal weight (Wa) divided by human weight (Wh). The LD50 of the aqueous leaf extract of Abrus precatorius in mice was estimated to be between 2559.5 and 3123.3 mg/kg body weight. The LD50 extrapolated from mouse to rat (1349.3-1646.6 mg/kg), hamster (1855.3-2264.1 mg/kg), guinea pig (1279.5-1561.4 mg/kg), rabbit (618.4-754.7 mg/kg), monkey (593.7-724.5 mg/kg), cat (392.7-479.2 mg/kg), dog and baboon (371.1-452.8 mg/kg), child (297-362 mg/kg) and adult human (197.8-241.5 mg/kg) body weight respectively could be a reality. The therapeutic safe dose range for the animals was 1-12.5 mg/kg body weight for a period of 7 days, but at a dose (≤ 200 mg/kg body weight) the leaf extract showed haematinic effect. However, at a higher dose (> 200 mg/kg), the extract showed haemolytic activity in rats, whereas at a dose (≥25.0 mg/kg), the leaf extract might be organotoxic in hamster, guinea pig, rabbit, monkey, cat, dog, baboon, child and adult human if administered orally for a period of 7 days.


INTRODUCTION
Abrus precatorius is highly regarded as a universal panacea in a herbal medicine (49). The aqueous leaf extract of Abrus precatorius showed 99.4% clearance of Plasmodium berghei in mice within 11 days (47), significant antimicrobial activity against Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Escherichia coli and Salmonella typhimurium (4,42,43). The plant showed significant inhibition of human metabolic breast cancer cell line (MDA-MB 231). The ethanolic extract of Abrus precatorius leaves may be used in the management of asthma (52). It also inhibited acetylcholine-induced contractions of both toad rectus abdominis and rat phrenic nervediaphragm muscle preparations and caused flaccid paralysis in the young chicks (56). Both natural and heat denatured forms of abrusagglutinin are potential immunomodulators (54).
The leaves are used to cure fever, stomatitis, bronchitis (29), epilepsy (6), neuronal damage (35) and diabetes (13). Two triterpernoid saponins isolated from aerial parts of Abrus precatorius exhibited activity against inflammation (5), gonorrhoea, diarrhea and dysentery (8). The ethanol acetate of the leaf extract showed anti-serotonergic activity in frog fundus strip (11). The leaves also have astringent, emetic, antihelmintic, alexeteric and diuretic properties in addition to being useful in cough (32), pharyngodynia, pectoralgia (28), strangury and vitiated condition of vata (25,29). Isoflavanquinone, abruquinone B and abruquinone G isolated from aerial parts of the plant exhibited antitubercular, antiplasmodial, antiviral and cytotoxic activities (30). The leaves are sweet tasting due to the presence of glycyrrhizin component. The leaves are also used for the treatment of scratches, sores and wounds caused by dogs, cats and mice. Fresh leaves may be pressed on the gum for the treatment of stomatitis, skin cancer and nervousness (8), suppuration, acne, boils, abscesses, tetanus, rabies (58), colic, convulsion (27), cough, sore throat and insomnia (33). Aqueous extract of the plant has haematinic and plasma expander effects in mice (38).
The clinical features of Abrus precatorius leaf poisoning were pulmonary oedema and hypertension (16). The aqueous extract of A. precatorius leaf caused decreased levels of packed cell volume, haemoglobin concentration, red blood cell count, white blood cell count, mean corpuscular volume and mean corpuscular haemoglobin concentration in rats. The extract also resulted in increased levels of total serum protein, albumin, alanine amino transferase, aspartate amino transferase, alkaline phosphatase and total bilirubin. It also caused testicular degeneration characterized by decreased numbers of epithelial lining and reduction in sperm cells at dose range between 400 and 1600 mg/kg body weight (3). The foliage of Abrus precatorius contains abrin, which is among the most potent toxins. Clinical toxicosis was characterized by gastroenteritis, weakness and death (2,10,20). Abrin is present in the leaf and is known to cause hyperactivity of physiological system (8). The parenteral LD 50 of abrin in mice is less than 0.1μg/kg (8). All parts of Abrus precatorius are toxic (9).
Acute toxicity study of aqueous extract of Abrus precatorius leaf in mice showed shallow respiration, sedation, weight loss, penile prolapsed and limbs paddling within 48 hours of extract administration. Haematological analysis revealed significant increased packed cell volume, red blood cell and white blood cell count (44). The biochemical analysis showed increased aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, chloride ion, hypophospataemia, hyponatraemia, hypokalaemia and hypoglycaemia after 3 weeks of oral administration of the extract at dose range between 25.0 and 200 mg/kg body weight. At 12.5 mg/kg only the urea increased on the 7 th day of the extract administration (46). In vitro cytotoxicity assay of Abrus leaf against rat myoblast (L 6 ) cell line showed that chloroform fraction was the most toxic with inhibitory concentration (IC 50 ) value of 43.7μg/ml followed by n-hexane fraction (44.3μg/ml). In vitro antiplasmodial assay against plasmodium chloroquiune-pyrimethanine resistant strain (K 1 ) showed that n-hexane fraction has the best activity with IC 50 value of 12.1μg/ml followed by chloroform fraction (23.0μg/ml) (40). The LD 50 of oral and intraperitoreal aqueous extract of Abrus precatorius leaf in mice were 2559.5 to 3123.2 mg/kg and 866 mg/kg body weight respectively (45). In view of the toxic potential of Abrus precatorius and also in line with the principle of replacement, reduction and refinement, the toxicity potential of the plant was extrapolated from mice to other fifteen species of monogastric animals.
Animal-human and human-animal dose translations of LD 50 , toxic and therapeutic doses of aqueous leaf extract of Abrus precatorius were determined using the human equivalent dose formula. Human Equivalent Dose (HED) is equal to animal dose multiplied by animal km factor divided by human K m factor. The K m factor is body weight (kg) divided by body surface area (m 2 ). Human equivalent no-observable adverse effects dose is equal to animal no-observable adverse effect level (NOAEL) multiplied by animal weight (Wa) divided by human weight (Wh) to the power of 0.33 was used to confirm 12.5 mg/kg body weight of mice (relatively safe dose) translated to human and other animals' safe doses (4,8,9,17,34,41,51,55 A safety factor between 10 th and 1000 th was used to determine ideal safe therapeutic doses.

Publications
Animal doses (mg/kg) Human equivalent doses (mg/kg) Saganuwan   Human-animal equivalent therapeutic dose translation showed that 1 mg/kg body weight of adult human translated to 1.1 mg/kg in mini-pig and 9.1 mg/kg body weight in hamster respectively. But, 1.9 mg/kg body weight in human translated to 2.02 mg/kg in mini-pig and 18.1 mg/kg in hamster respectively. However, 15.5 mg/kg in human translated to 16.6 mg/kg in micro-pig and 145.0 mg/kg in hamster (Table 3). Human to hamster, rat, guinea-pig, rabbit, monkey, cat, baboon, ferret,   Table 3. At dose range between 400 and 1600 mg/kg body weight reported by Adedapo et al. (3), there was decreased RBC, PCV and Hb which were increased in mice (40,44). White blood cells and lymphocytes were decreased in rat (3) ( Table 4).
Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, protein, albumin, globulin, albumin/globulin ratio were reported to have increased in rats and mice (3,46). Total bilirubin, direct bilirubin and indirect bilirubin were reported to have increased in rat (3) but decreased in mice (46). Urea, creatinine, chloride ion, calcium ion and glucose increased as sodium ion  (Table 5).

DISCUSSION
The mouse-human translated LD 50 of 197.9-241.5 mg/kg body weight agrees with the report of Yamba et al. (58) indicating that the human translated LD 50 should be between 10 th and 100 th of LD 50 in mice. This shows that adult human is the most sensitive to acute poisoning of Abrus precatorius leaf followed by child, mini-pig, micro-pig, baboon, dog, cat, monkey, rabbit guinea pig, rat, hamster and mouse in that order (Table 1) invariably rating the extract as slightly to moderately toxic in monogastric animals (26,40,41). But the calculated higher LD 50 of child (297 -362.3 mg/kg) in comparison with the LD 50 of adult human (197.9 -241.5 mg/kg) may be caused by high body surface area of the child in comparison with that of the adult. Hence the child may require more amount of the extract than the adult. Generally the child weighing 20 kg should have developed fully metabolic enzymes. High body surface area is given low km. Connotatively, fatty individuals may be less susceptible to Abrus precatorius poisoning than lean individuals. However 12.5 mg/kg body weight that translated to 1.0 mg/kg showed no observable adverse effect when administered for a period of 3 days to Plasmodium berghei infected mice, although the dose cleared 98.7% of the parasites in the blood within 9 days after the extract treatment (47). These findings agree with the report of Saganuwan (40) indicating that the Nupe ethnic group from Bida emirate has been using Abrus precatorius leaf for the treatment of both acute and chronic malarial symptoms. The use of Abrus leaf in the treatment of malaria among Nupes is sometimes either the last option after the conventional antimalarial drugs might have failed or due to poverty. The administration of 12.5 mg /kg body weight of the extract for 7 days did not cause any observable adverse effect on the treated mice but caused slight increase in the plasma urea level (37,45). Therefore 1 mg/kg body weight translated dose for a period of 3 days may be safe in human, but when administered for a period of 7 days may likely cause slight increased plasma urea in human. Hence, one-tenth safety factor of mouse therapeutic dose can be adopted in human, whereas the 100 th to 1000 th safety factor of Abrus precatorius leaf extract may be adopted for doses between 10 and 100 mg/kg body weight of the extract. The least sensitive animal to Abrus precatorius aqueous leaf extract in this study may be mice, followed by hamster, rat and guinea pig respectively. However, doses higher than 1.5 mg/kg may be toxic to a child. Also doses higher than 9.1, 6.6, 6.5, 3.0, 2.9, 1.9, 1.8, 1.8, 5.4, 6.5, 5.6, 1.4 and 1.1 mg/kg body weight may be toxic to hamster, rat, guinea pig, rabbit, monkey, cat, dog, baboon, ferret, squirrel monkey, micro-pig and minipig respectively (Table 3). Animal human equivalent dose projections can be done in three ways; doses expressed in mg/kg body weight for the species where "the critical effect" leading to "the reference dose" are adjusted to mg/kg body weight 0.75 to reflect the dependence of pharmacokinetic elimination on metabolic rate-which may tend to scale with (body weight) 0.75 (1,34,53). Although when we scaled the body weight to 0.75 the estimated LD 50s were seriously higher in comparison with 0.67. A further adjustment factor may be applied to refl ect the median human maximum tolerated dose (MTD) expected based on the identity and number of species that provided data that potentially could have been used as the basis for the reference dose (24). The expected geometric means of the ratios of human toxic potency ( ) to the toxic potency estimated from the animal experiments ( ) or ( ) are based on the ratios available for each species or combination of species shown (15). Looking from the single-species analyses to the cases for which data for increasing numbers of species are available for choice of "the most sensitive", it can be seen that the geometric mean ratios of the observed human potency to the human potency projected from the animal potency per (body weight) 0.75 tend to decline. This is the natural result of the fact that the lowest toxic dose inferred for data for more species will tend to make a more "conservative" prediction of human potency than when data are only available for a single animal species. Finally, the uncertainty in each type of animal-to-human toxic potency projection is inferred from variability of the ratios of the observed human potencies to the animalprojected potencies for different compounds. These variabilities are modelled as lognormal distributions with the standard deviations of the logarithms of the observed human to animal-projected potency ratios (24).
Increased RBC, PCV and haematocrit at dose range between 25-200 mg/kg body weight showed that the plant aqueous leaf extract, have haematinic effect at lower dose levels (44). But at higher dose levels (400-1600 mg/kg), it caused haemolysis with resultant hyperbilirubinaemia (3). However, at both lower doses (25-200 mg/kg) and higher doses (400-1600 mg/kg) the plant leaf extract caused increase in biochemical parameters with resultant deleterious effect on kidney, heart, intestine, lung, spleen and liver. But Adedapo et al. (3) should have conducted acute toxicity study on rats to serve as a guide for their selection of therapeutic doses instead of using predetermined doses. More so, their histopathological studies were restricted to testes, kidney and liver bearing in mind the fact that the plant is organotoxic. When Saganuwan and Onyeyili (40) treated mice with 25-200 mg/kg body weight of Abrus leaf extract for 21 days, the extract caused death of some mice, most especially at higher doses (25 -200 mg/kg). But, it looked strange that Adedapo et al. (3) didn't report death in their experimental animals with serious kidney and liver damage. The plant has been reported to contain toxalbumin (phytoprotein), abrin, which may be responsible for the observed toxicity effects (21). Abrin consists of abrus agglutinin, and toxic lectins [a] to [d], the five toxic glycoproteins found in the plant. Abrus agglutinin is non-toxic to animal cells but a potent haemaglutinator (7). The toxic portion of abrin is heat-stable to incubation at 60 0 C for 30 minutes, but at 80 o C most of the toxicity is lost in 30 minutes (36). Although the plant, particularly the seed is known to be highly poisonous due to presence of abrin (7,12,14,21,36), the leaf in the present study was observed to be slightly toxic. The incubation of the abrus leaf extract at 60 o C for several hours must have reduced the toxicity of the leaf (46). Although the extract was administered orally, abrin is very stable in the gastrointestinal tract, from where it is slowly absorbed and thereby making it less toxic (19). Abrins's toxic effect is due to its direct action on the membrane of parenchymal cells (e.g. liver, kidney cells and erythrocytes) (23) via the B chain (haptomere) that binds to galactosyl-terminated receptors on the cell membrane, which is a prerequisite for the entry of the other subunit, the A chain (effectomere). This inactivates the ribosomes, arrests protein synthesis, and causes cell death (50). The A-chain attacks the 60s subunits of the ribosomes and by cutting out elongation EF 2 stops synthesis of protein (18). So the extraction and concentration of aqueous extract of Abrus precatorius leaf at 60 o C (46) and unknown temperature (3) must have reduced toxic effects of the leaf extract in mice and rats respectively.

CONCLUSION
The translated mouse-human LD 50 is 197.9-241.5 mg/kg body weight, showing high level of sensitivity of humans to Abrus precatorius leaf poisoning. The safe human therapeutic dose for a period of 3 days may be 1.0 mg/kg body weight. But the safe therapeutic doses for other animals are; hamster (9.1 mg/kg), rat (6.6 mg/kg), guinea pig (6.5 mg/kg), rabbit (3.0 mg/kg), monkey (2.9 mg/kg), cat (1.9 mg/kg), dog and baboon (1.8 mg/kg) and child (1.5 mg/kg) respectively. However, the administration of the safe doses for a period of 7 days may cause slight increase in the plasma urea.