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Comparative Potential Therapeutic Effect of Sesame Oil and Peanut Oil against Acute Monocrotaline (Crotalaria) Poisoning in a Rat Model

Authors


Corresponding author: Ming-Yie Liu, PhD, Department of Environmental and Occupational Health, National Cheng Kung University College of Medicine, 138 Sheng-Li Road, Tainan 70428, Taiwan; e-mail: myliu@mail.ncku.edu.tw.

Abstract

Background

Many Crotalaria plant species contain hepatotoxic pyrrolizidine alkaloids (such as monocrotaline) that can cause acute and chronic poisoning in cattle and other animals.

Hypothesis

Peanut oil, atropine sulfate, and antidiarrheal agents are used to treat acute monocrotaline poisoning. The effect of sesame on acute monocrotaline poisoning has never been investigated.

Animals

Fifty male Sprague-Dawley rats were used for toxicity studies.

Methods

Experiment 1: Group I, control. Groups II–IV were given monocrotaline (205.2 mg/kg) and euthanized 6, 12, and 24 hours later. Experiment 2: Group I, control. Group II monocrotaline alone (205.2 mg/kg). Groups III–VI were given monocrotaline (205.2 mg/kg) and 1 hour later, Groups III and IV were given sesame oil (1 and 2 mL/kg) and Groups V and VI were given peanut oil (1 and 2 mL/kg).

Results

Monocrotaline significantly decreased (< .05) serum amylase activity, but, over time, increased (< .05) pancreatic and lung injury. AST and ALT activity and liver injury peaked at 24 hours. Sesame oil and peanut oil (< .05) inhibited the changes in all tested parameters in acute monocrotaline poisoning. Although peanut oil inhibited acute monocrotaline poisoning, it induced steatosis, but sesame oil did not.

Conclusion and Clinical Importance

We hypothesize that early pancreatic and lung injury and late liver injury contribute to acute monocrotaline poisoning and that sesame oil is more efficacious than peanut oil against acute monocrotaline poisoning in rats. However, additional studies are needed to confirm that these oils have the same effects in cattle and other animals.

Abbreviations
ALT

alanine transaminase

AST

aspartate transaminase

Approximately 600 species of Crotalaria (family Fabaceae; rattle pods) grow in tropical and subtropical regions of the world.[1] Several species of Crotalaria are sources of the hepatotoxic pyrrolizidine alkaloid (PA) monocrotaline,[2] which sporadically poisons grazing horses, cattle, sheep, and pigs, as well as poultry whose feed grain is contaminated with their seeds.[3-6] The acute poisoning and death of cattle occur because of a large intake of Crotalaria over a short period under conditions of natural grazing. Acute poisoning is characterized by extensive damage, necrosis, and hemorrhage of the liver,[7, 8] which is the only abnormality recorded.[7] PAs are monoesters and diesters of the pyrrolizidine necine bases retonecine and otonecine from a variety of plant species, with 1,2-unsaturation of the necine base required for hepatoxicity.[9-11] New reports on PA plant toxicity continue to be recorded from various parts of the world in the veterinary and other scientific literature.[7] Nonetheless, Crotalaria poisoning is underreported.[12] Various diet supplement treatments are ineffective against PA intoxication in livestock.[12-15] Poisoned animals with clinical signs rarely recover; therefore, prevention is the best control measure.[15] Veterinary teams usually use local remedies for Crotalaria poisoning, for example, peanut oil, atropine sulfate, and antidiarrheal agents.[12]

Sesame oil, from the seeds of Sesamum indicum, protects against multiple organ failure,[16] sepsis,[17] hepatic injury after cecal ligation and puncture,[18] endotoxemic renal injury,[19] septic hepatic injury,[20] and acetaminophen-induced acute hepatic damage.[21] Sesame oil as a dietary supplement and nutraceutical alleviates many types of diseases in rodent models.[22-24] Sesame oil contains sesamin, sesamol, and sesamolin, all of which contribute to its antioxidant property.[25] Sesame oil offers better protection than other dietary oils, such as peanut oil, against hypertension, hyperlipidemia, and lipid peroxidation by modulating in vivo antioxidants.[26] Furthermore, sesamol, an active ingredient of sesame oil, therapeutically attenuates monocrotaline-induced acute liver damage.[27] Therefore, monocrotaline poisoning in rats is used as a model for similar poisoning in cattle. We hypothesized that sesame oil would prevent acute monocrotaline poisoning in rats. In this study, we compared the therapeutic effects of sesame oil and peanut oil on acute monocrotaline poisoning in rats.

Materials and Methods

Animals

Male Sprague-Dawley rats 7–8 weeks old and weighing 200 ± 50 g were purchased from our Institutional Laboratory Animal Center. They were given pellet feed1 and water ad libitum. They had a 12-hour light/dark cycle and central air conditioning (25°C, 70% humidity) throughout the experiment. The animal care and experimental protocols were in accordance with nationally approved guidelines (No. 99054).

Chemicals

Monocrotaline, sesame oil, and peanut oil were purchased from Sigma-Aldrich.2 To prepare a solution of monocrotaline (10 mg/mL), 500 mg of monocrotaline was completely dissolved in a minimal volume (3–5 mL) of 1.0 N HCl, then 40 mL phosphate-buffered saline (PBS) (pH 7.4) was added and the pH was adjusted to 7.4 with 0.5 N NaOH. The total volume was brought to 50 mL with PBS.[28] All other reagents were purchased from Sigma-Aldrich.

Experimental Protocol

Experiment 1

The rats were divided into 4 groups (n = 5 per group). Group I rats were untreated healthy controls. Group II, III, and IV rats were given monocrotaline (205.2 mg/kg)[29] PO and euthanized 6, 12, and 24 hours later, respectively. Serum amylase, AST, and ALT activity were assessed, and a small piece of pancreas, lung, and liver tissue from each rat was harvested and fixed in formalin for histopathologic examination.

Experiment 2

The rats were divided into 6 groups (n = 5 per group). Group I rats were untreated healthy controls. Group II rats were given only monocrotaline (205.2 mg/kg) PO. Group III, IV, V, and VI rats were given monocrotaline (205.2 mg/kg) PO and then, 1 hour later, Group III and IV rats were given sesame oil (1 and 2 mL/kg, respectively) PO and Group V and VI rats were given peanut oil (1 and 2 mL/kg, respectively) PO. After 24 hours, the activity of serum amylase, AST, and ALT was assessed, animals were euthanized, and approximately 1 cm3 of pancreas, lung, and liver tissue from each rat was harvested and fixed in formalin for histopathologic examination.

Collecting Blood

Blood was collected (after 0, 6, 12, and 24 hours, respectively, in Experiment 1 and after 24 hours in Experiment 2) in serum separation tubes by femoral venipuncture while the rats were under mild ether anesthesia. The tubes were left at room temperature for 30 minutes to clot and then centrifuged at 1000 × g at 4°C for 10 minutes.

Assessing Acute Poisoning

Pancreatic and hepatic damage was assessed by measuring the amylase, AST, and ALT activity in serum with a biochemistry analyzer.3 Pancreatic, lung, and hepatic injury was assessed by means of histological studies. Approximately 1 cm3 of tissue from each rat was cut and placed in 4% phosphate-buffered formalin. The tissue pieces were dehydrated with a graded percentage of alcohol and then fixed in paraffin wax for 1 hour to form blocks. The blocks were trimmed and cut into 4-μm-thick sections, stained with hematoxylin and eosin, and then mounted with Depex-Polystyrene dissolved in xylene mountant. The permanently mounted sections of tissue were examined under a microscope4 (magnification, 100×) to assess injury.

Histology and Scoring System

An average of 5 sections per rat was evaluated at both high- and low-power views. Scoring was done by a slightly modified protocol.[30] Numerical scores of 1–5 were based on the percentage of tissue affected: 1 = 0%, 2 = 1–25%, 3 = 26–50%, 4 = 51–75%, and 5 = 76–100%. Categories for lung damage (changes) included interstitial changes (interstitial or interalveolar septal thickening), inflammation (intra-alveolar neutrophilic infiltrate), and consolidation (a combination of both cellular debris and fibrin-filled alveolar space).[30] For pancreas-based inflammation, the percentage of damage to the islets of Langerhans and acini was scored as 1 = 0%, 2 = 1–25%, 3 = 26–50%, 4 = 51–75%, and 5 = 76–100%. Liver steatosis was scored by a slightly modified protocol.[31, 32] A score of 1 was assigned to normal tissue. Numerical values of 2–5 were assigned depending on the percentage of clear vacuoles that contained lipid: 1 = 0%, 2 = 1–25%, 3 = 26–50%, 4 = 51–75%, and 5 = 76–100%.[27]

Statistical Analysis

The statistical analysis was done by SPSS 11.0.1.5 Data are presented as means ± SD (standard deviation). Differences in the measured variables between groups were assessed by a Fisher's Least Significant Difference test. Significance was set at < .05.

Results

Experiment 1

Acute Monocrotaline Poisoning of the Pancreas

Serum amylase activity was significantly lower in Groups II–IV than that in Group I (0 hour; range, 2000–3500 IU/L) (Group II [6 hours] [< .05]; Groups III [12 hours] and IV [24 hours] [both < .01]) (Fig 1A). Pancreatic histology from Group II showed severe vacuolization and loss of islets of Langerhans. Group III showed an extensive loss of islets of Langerhans with necrosis. Group IV showed extensive loss of islets of Langerhans, necrosis, and hemorrhage in the remaining islets (Fig 1B and C).

Figure 1.

Effect of acute monocrotaline poisoning on serum amylase and the pancreas. Group I (n = 5) rats were healthy controls. Group II, III, and IV (n = 5 each) rats were gavaged with monocrotaline (205.2 mg/kg) and euthanized after 6, 12, and 24 hours, respectively. (A) Serum amylase, (B) pancreatic histology, and (C) pancreatic histological scoring. Photomicrographs were taken at 40×. Insets show vacuoles and degranulated islets. Data are means ± standard deviation (SD).a,b,c The differences between treatments with different letters are significant (< .05).

Acute Monocrotaline Poisoning of the Lungs

There was more histological evidence of lung injury in Groups II, III, and IV than in Group I (Fig 2A), with severe hemorrhage, interstitial thickening, and cellular infiltration in the interstitium and alveolar compartments. In addition, Group III showed greater interstitial thickening, and Group IV showed thickening of the bronchial cartilage. Lung histological scores were significantly higher (< .05) in Groups II–IV than in Group I (Fig 2B).

Figure 2.

Effect of acute monocrotaline poisoning on the lungs. For group treatment information, please see the Figure 1 legend. (A) Histopathological sections and (B) histological scoring. Photomicrographs were taken at 40×. Insets show severe hemorrhage and interstitial thickening. Data are means ± SD. a,bThe differences between treatments with different letters are significant (< .05).

Acute Monocrotaline Poisoning of the Liver

Serum AST and ALT activity were significantly (< .05) higher in Group IV than in Groups I (AST; range, 50–80 IU/L and ALT; range, 25–45 IU/L), II, and III (Fig 3A and B). Liver histopathology from Groups II and III showed mild changes in sinusoids. However, Group IV showed severe hepatic necrosis with rounding up of sinusoidal endothelial cells, congestion, sinusoidal alteration, destruction of the sinusoidal lining, and severe hemorrhage (Fig 3C and D).

Figure 3.

Effect of acute monocrotaline poisoning on liver markers. For group treatment information, please see the Figure 2 legend. (A) Aspartate transaminase (AST), (B) alanine transaminase (ALT), (C) liver histology, and (D) liver histology scoring. Photomicrographs were taken at 40×. Insets show hepatic necrosis and red blood cell penetration in the sinusoid. Data are means ± SD.a,b,c The differences between treatments with different letters are significant (< .05).

Experiment 2

Effect of Sesame Oil and Peanut Oil on Pancreatic, Lung, and Liver Injury

Serum amylase activity was significantly higher in Groups III and V (treated with monocrotaline and 1 mL/kg of sesame oil or peanut oil) than in Group II (treated with monocrotaline only) (Fig 4A). However, there were no significant differences in Groups IV and VI (treated with monocrotaline and 2 mL/kg of sesame oil or peanut oil) compared with Group II. Pancreatic pathology, however, showed significant differences in Groups III–VI compared with Group II (Fig 4B and C).

Figure 4.

Effect of sesame oil and peanut oil on serum amylase and the pancreas in acute monocrotaline poisoning in rats. Group I (n = 5) rats were healthy controls. Group II (n = 5) rats were gavaged with monocrotaline (205.2 mg/kg). Group III and IV (n = 5 each) rats were gavaged with monocrotaline (205.2 mg/kg) and, 1 hour later, gavaged with sesame oil (1 and 2 mL/kg, respectively). Group V and VI (n = 5 each) rats were gavaged with monocrotaline (205.2 mg/kg) and, 1 hour later, gavaged with peanut oil (1 and 2 mL/kg, respectively). (A) Serum amylase, (B) pancreatic histology, and (C) pancreatic histology scoring. Photomicrographs were taken at 40×. Data are means ± SD. a,b,c,dThe differences between treatments with different letters are significant (< .05).

Lung pathology from Groups III and V showed a marked decrease in hemorrhage, interstitial thickening, and mild cellular infiltration in the interstitium and alveolar compartments compared with Group II (Fig 5A). Lung histological scores were significantly lower (< .05) in Groups III–VI than in Group II (Fig 5B).

Figure 5.

Effect of sesame oil and peanut oil on lung injury in acute monocrotaline poisoning in rats. For group treatment information, please see the Figure 4 legend. (A) Histopathological sections and (B) histological scoring. Photomicrographs were taken at 40×. Data are means ± SD.a,b,c,d The differences between treatments with different letters are significant (< .05).

Serum AST and ALT activity was significantly (< .05) decreased in Groups III–VI compared with Group II (Fig 6A and B). Liver histopathology from Groups III and IV showed little necrosis, few mild alterations in sinusoidal endothelial cells, minor changes in sinusoidal lining, and no hemorrhage compared with Group II. Most of the liver sections from Groups V and VI showed more severe steatosis than did those from Groups III and IV (Fig 6C). Steatosis scores were significantly higher in Groups II, V, and VI than in Groups I, III, and IV. Sesame oil efficiently decreased steatosis in Groups III and IV. However, peanut oil had no effect against steatosis in Groups V and VI compared with Group II (Fig 6D).

Figure 6.

Effect of sesame oil and peanut oil on liver injury in acute monocrotaline poisoning in rats. For group treatment information, please see the Figure 4 legend. (A) Aspartate transaminase (AST), (B) alanine transaminase (ALT), (C) liver histology, and (D) steatosis score. Data are means ± SD.a,b,c,d The differences between treatments with different letters are significant (< .05).

Discussion

We found that early pancreatic and lung injury and then late liver injury contributed to acute monocrotaline poisoning in rats. Acute ovine intoxication from a single dose of Crotalaria retusa seeds (205.2 mg of monocrotaline/kg body weight) caused mild jaundice and the death of sheep within 12 hours.[29, 33] Necropsy and histology of the liver disclosed a nutmeg appearance, centrilobular necrosis, and hepatic periacinar necrosis.[29, 33] We treated all the rats with the same dose of monocrotaline, and the rats were all able to survive to 24-hour post poisoning with no mortality during this experiment. However, we found that serum amylase activity had decreased and that severe pancreatic and lung injury had developed within 6 hours, but that serum AST and ALT activity and hepatic injury had peaked 24 hours after acute monocrotaline poisoning. Therefore, we hypothesize that the death of the sheep in Anjos et al[29] and Nobre et al[33] may have been because of early pancreatic and lung injury along with late liver injury in acute intoxication by Crotalaria retusa.

Sesame oil and peanut oil attenuated acute monocrotaline poisoning in our rat model. Peanut oil has been used as a local remedy for Crotalaria poisoning in cattle.[12] Sesame oil and peanut oil attenuated pancreatic, lung, and liver injury in acute monocrotaline poisoning. Sesame oil efficiently decreased steatosis, but peanut oil was ineffective. Steatosis is associated with long-term complications and risk factors, including diabetes mellitus,[34] protein malnutrition,[35] hypertension,[36] and obesity[37] in rats and humans; these conditions have profound adverse effects on the pancreas, liver, and heart. We conclude that sesame oil is more beneficial than peanut oil in attenuating multiple organ injury in acute Crotalaria poisoning. Furthermore, sesame oil is effective against drug- and chemical-induced organ injury.[38-42] Sesame oil contains palmitic acid (8.58%), stearic acid (5.44%), and acids with a small amount of arachidic acid (0.9%), and its main unsaturated fatty acids are linoleic acid (46.26%) and oleic acid (38.84%).[43] In addition, it contains phenol, sesamin, sesamol, sesamolin, and a relatively small amount of tocopherol, which contributes to its oxidative stability.[25] In contrast, peanut oil is composed of approximately 80% unsaturated fatty acids with palmitic acid (7–11%), stearic acid (2–4%), oleic acid (48–54%), linoleic acid (27–38%), linolenic acid (0.62–1.12%), and arachidic acid (1.44–2.36%).[44] Sesamol is a phenolic nonfat potent antioxidant; its rapid absorption (within 1 hour), distribution and metabolism (1 hour), and elimination (4–8 hours) without accumulation[45-47] indicate its versatile nature for prophylactic and therapeutic use against various toxic models in rodents. Sesamol prophylactically and therapeutically attenuates multiple organ injury in various disease models.[27, 48-50] Whether or not SC injected sesamol is useful in attenuating acute Crotalaria poisoning in cattle still is unknown. More investigation is needed.

Sesame oil is known to be harmless when taken in recommended dosages against oxidative stress-associated hepatic injury after cecal ligation and puncture in rats.[20] When taken in a large dose, it does not have a cumulative antioxidative effect; instead its antioxidative effect is decreased.[20] In addition, dietary fat with polyunsaturated fatty acid contributes to steatohepatitis.[51] Hepatic metabolism of fat[52, 53] and monocrotaline[32] requires cytochrome P-450 isozymes. A higher dose of oral sesame oil and peanut oil may contribute to steatohepatitis as well as affect metabolism and elimination of monocrotaline. An increased burden on liver metabolism as well as overall stress may be the reason for the increases in ALT and AST activity in Group IV and VI rats.

The implication of the present study is that it can be extrapolated to cattle pyrrolizidine alkaloid toxicosis. However, additional studies are needed. In addition, no definitive treatment for pyrrolizidine alkaloid toxicosis exists. Treatment consists of supportive care to allow time for liver regeneration and restoration of liver function. Supportive care includes administering IV fluid to correct dehydration and electrolyte abnormalities, glucose to provide basic energy requirements, and antibiotics to prevent infection. Local remedies include certain herbs. Veterinary teams that consist of a veterinarian, credentialed veterinary technician or veterinary technologist specialist, veterinary assistant, and certified veterinary practice manager usually administer groundnut or palm oil, atropine sulfate, or other antidiarrheal agents.[12, 54-56] Ruminants have 4-chamber stomachs with strong digestive and absorptive power. The abomasal compartment corresponds to the stomach of the nonruminant, and it secretes gastric juice with a pH range from 2.0 to 2.5. This low pH may facilitate breakdown of fatty acids in sesame oil and enhance absorption when given PO. Prophylactic and therapeutic SC injection of sesame oil attenuates acute renal injury in mice model.[57] Subcutaneous injection of sesame oil may be effective in treating organ injury in cattle. Sesame oil was therapeutically effective for treating pancreatic, lung, and liver injury caused by monocrotaline poisoning in our rat model. Despite sesame oil's potent therapeutic effect against acute monocrotaline poisoning in rats, its use in cattle requires additional investigation.

Conclusion

We hypothesize that early pancreatic and lung injury along with late liver injury contribute to acute monocrotaline poisoning. Sesame oil was a more efficacious therapeutic agent than peanut oil for treating acute monocrotaline poisoning. However, additional investigation is needed.

Acknowledgments

This research was supported by grants NSC 99-2314-B-006-031-MY3 and NSC 99-2221-E-006-064-MY3 from the Taiwan National Science Council.

Conflict of Interest

None of the authors of this article has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the article. We declare that we have no conflicts of interest.

Footnotes

  1. 1

    Richmond Standard; PMI Feeds, Inc, St. Louis, MO

  2. 2

    St. Louis, MO

  3. 3

    Fujifilm Dri-Chem 3500s; Fujifilm, Kanagawa, Japan

  4. 4

    Eclipse E 600; Nikon Instech Co, Ltd, Kawasaki, Japan

  5. 5

    SPSS Inc, Chicago, IL

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