Abstract
Introduction
Aluminum phosphide (ALP) is a solid pesticide commonly applied for preserving rice and grain. In Iran it is known as the rice tablet which is cheap and highly toxic. 1 After exposure to moisture it releases phosphine gas (PH3), the active pesticide component with garlic-like odor, which is immediately absorbed through inhalation, ingestion or contact. With oral intake, the phosphine gas released is absorbed by the gastrointestinal tract by simple diffusion and is mainly excreted by the kidneys and lungs. 1,2 This gas is thought to have the central role in mechanism of ALP toxicity as it inhibits cytochrome c oxidase. 3 There are many reports of serious phosphide poisoning including fatalities from India and other developing countries. 4,5 For example, for a decade it has become the most common chemical for unintentional poisoning in India. 6 –9 In a study of 217 poisoned children in Northern India, 58% of all ALP-intoxicated patients died and this agent accounted for 62% of all deaths. 10 It is a rare way for suicide except in Iran and Jordan from where a few case series have been reported. 11,12 In a retrospective 7-year study of ALP poisoning in Tehran, 471 patients were admitted to a referral hospital with ALP poisoning. In that study, the overall case fatality ratio was 31%. 13 Deaths which usually occur within 12–24 hours are secondary to peripheral vascular collapse, cardiac failure and adult respiratory distress syndrome (ARDS). Inhibition of plasma cholinesterase activity and also methemoglobinemia has been reported from animal studies in acute ALP poisoning. 14 –18 By diminution of oxidant stress caused by phosphine, trimetazidine is used for the treatment of cardiac toxicity. 18 Some animal studies have suggested increased survival time with the use of N-acetylcysteine and pralidoxime. 15,19 There is no antidote to phosphine or ALP poisoning and a great majority of patients die despite intensive care. Thus, supportive measures are all that can be offered. 20 In vitro experiments revealed that vegetable oils can inhibit the release of phosphine from ALP. 21 Non availability of specific antidote, and also limitation of supportive care strategies have been our reasons for conducting the present study. In this study the effect of sweet almond oil on ALP toxicity was evaluated and compared with control group in an animal model. Consistent with the management protocols of patients with toxicological emergencies, there is a necessity to study various diagnostic and ancillary tests; therefore, the plasma activity of cholinesterase was analyzed to evaluate the efficacy of intervention.
Methods
Experimental
Adult Wistar strain rats (n = 35) of either sex were purchased from a private animal house, all weighing between 200 and 250 grams. They were acclimatized for 1 week under controlled conditions in which a 12 h/12 h light/dark cycle was maintained in a temporary animal house with access to food and water ad libitum. The experiments performed in this study have been carried out according to the World Medical Association statement on animal use in biomedical research. 22 Exposure to ALP and experiments assessing the effects of ALP on rats were carried out in a separate, isolated laboratory under the same environmental condition as the colony room. The animals were divided into four groups: group 1 (control group, n = 5; saline treated, intragastric [IG]), group 2 (n = 10; ALP exposed, 11.5 mg/kg ALP, IG), group 3 (n = 10; ALP exposed, 11.5 mg/kg ALP + 10 ml sweet almond oil, IG) and group 4 (n = 10; ALP exposed, 11.5 mg/kg ALP + 10 ml sweet almond oil 30 min later, IG). The rats in group 1 were sham operated and saline treated. In groups 2–4, rats were monitored for survival times. The animals were fasted overnight with free access to water before experiment. In all animals, anesthesia was induced by injecting pentobarbital (30 mg/kg, intraperitoneal) then a vertical incision was made over epigastrium. By exposing the stomach, stomach was occluded by knotting suture material around its upper portion and powdered ALP in normal saline was administered into the stomach through a catheter. In group 3, 10 ml sweet almond oil was administered immediately, and after withdrawal of catheter, gastrostomy hole was closed by tightening sutures. In group 4 the same steps were repeated, except that nelaton catheter was hold in place by purse suture followed by sweet almond oil administration 30 minutes later. Besides measuring plasma cholinesterase in control group, plasma cholinesterase was measured 30 minutes after ALP administration in groups 2 and 3, and 60 minutes later in group 4. Just before gaining consciousness the incision was closed. The animals were then monitored for evaluation of health status for 1 week as a recovery phase.
The samples for cholinesterase activity were collected into test tubes and centrifuged (4500 rpm for 5 min) at room temperature. The supernatants were separated and were stored at −20°C until being analyzed. Cholinesterase activity in the supernatant was measured spectrophotometrically at 405 nm using an auto analyzer (Hitachi-902, Hitachinaka, Japan) following Pars Azmun kit instructions.
Drugs
ALP (phostoxin, 3 mg tablet, ALP 56%, w/w) was from Shanghai AgroChina International Co. Ltd., China. Sweet almond oil, 60 ml/bottle, Kimia Pack Co., Karaj, Iran.
Statistics
The survival times of different groups were compared using Log Rank method of Kaplan-Meier analysis. The p value of less than 0.05 was considered significant. All analyses were done using SPSS V.16. Survival times of different groups were noted using estimated means (minutes) ±standard error (SEM).
Results
All the animals in group 1 survived and all in group 2 died. The mean survival time of rats exposed to ALP without any intervention was 101.1 ± 1.4 minutes (min). In the intoxicated rats which were given sweet almond oil immediately after poisoning (group 3), 4 rats survived and among the dead ones only one could live for 22 hours, with others being dead with maximum survival time of 200 min. The mean survival time for rats of group 3 was 4250.50 ± 1508 min. In group 4, the one with delayed intervention, the mean survival time was 1364.10 ± 933.03 min and one rat survived. Here in group 4, among 9 dead rats, 2 animals could live for 23 and 24 hours with others being dead of which the maximum survival time was 120 min (Figure 1 ). In analysis of survival times, There was a significant difference between group 2 which received ALP and the groups which underwent intervention (groups 2 and 3, p < 0.001; groups 2 and 4, p = 0.007). We also found that the mean survival time in animals received oil immediately, was markedly higher than the group 4 with delayed intervention (p = 0.049).
Survival curves of four study groups.
Mean cholinesterase levels (units/liter; U/L) were measured; group 1: 760 ± 32 U/L; group 2: 425 ± 30 U/L; group 3: 650 ± 30 U/L; group 4: 470 ± 20 U/L. Significant reduction in plasma cholinesterase activity for about 45% in group 2 and about 40% in group 4 were observed (Mann-Whitney U test, p < 0.01). There was no significant difference between inhibition of cholinesterase activity in groups 2 and 4 (Mann-Whitney U test, p = 0.191). On the other hand, the difference between reduced levels of cholinesterase were significant in groups 2 and 3 (Mann-Whitney U test, p = 0.001) and in groups 3 and 4 (Mann-Whitney U test, p = 0.039). During observing the animals we saw over secretion in nose and airway passages as a common presentation in some rats which may reflect the cholinergic effects of phosphine.
Discussion
The present study has demonstrated that intragastric sweet almond oil could increase survival time of animals exposed to ALP. There have been a few case reports of survival in patients of ALP poisoning when they were treated with vegetable oils especially with coconut oil. 21,23 In one human exposure to 12 g oral ALP, coconut oil was used with favorable outcomes. 23 In one study it has been shown that the survivors following ALP ingestion had more severe vomiting and lower hypotension and metabolic acidosis than the nonsurvivors. 24 This indirectly implies the importance of decreasing the burden of chemicals and the rapid rate of gastric absorption in this kind of toxicity. As seen in the present study, the poisoned animals undergoing immediate intervention with sweet almond oil had better outcomes considering both mean survival time and complete survival rate. Although the role of oils in providing better outcomes in patients has been shown through previous studies, their mechanisms of action were not clear. 23
One possible mechanism of toxicity with ALP is inhibition of cholinesterase activity; Works by Potter et al. 25 and Pazynich et al. 26 showed that phosphine can inhibit cholinesterase. Phosphine is mainly consumed as an intermediary in organophosphorus chemistry. Work by Lawson et al. 27 suggested that phosphine or phosphine-derived chemicals may be working through production of organophosphines. For the first time, Mittra et al. 28 showed 40% survival rate of animals exposed to ALP by using atropine and pralidoxime chloride. They concluded cholinesterase inhibition as one of the underlying mechanisms for ALP intoxication. Noting a variety of survival times in our study reflects the presence of unknown mechanisms for ALP intoxication. As mentioned above in our study results, despite significant inhibition of cholinesterase activity (40%) in group 4 (p < 0.01), the difference between survival times in groups 2 and 4 was significant (p = 0.007) and survival rate was 10% compared to no survival in group 2 with shorter mean survival time of about 101.1 ± 1.4 min. Interestingly, it is obvious that although there was no significant difference between levels of plasma cholinesterase in these two groups (p = 0.191), we found favorable outcomes in the group which received ALP with sweet almond oil 30 min later (group 4). It should be noted that the reduction rate of plasma cholinesterase in group 3 was lower than the groups 2 and 4 and the difference was significant (group 2 and 3, p = 0.001; group 3 and 4, p = 0.039).Pointing at these measures indicates that the timing of intervention with almond oil may have a role in gastric phase of phosphine absorption. On the other hand, noting the mortality rate of about 60% in group 3 despite no significant decrease in cholinesterase activity implies that unknown mechanisms other than cholinesterase inhibition for phosphine poisoning should be taken into account. Although serum cholinesterase activity was measured in this study as the only possible and available laboratory marker, there was no intention to use the results for establishing the cause and effect relationship. However, there is no clear explanation why some animals had favorable outcomes despite significant reduction of cholinesterase activity and some animals with normal cholinesterase activity had disappointing outcomes. Oils may possibly act through different mechanisms: decreasing the aqueous fraction of gastric juice or covering the surface of chemicals and preventing their activation, or interfering with the enteral phase of metabolism or possible innate characteristics of one specific oil (its antioxidant role or effect on mucosal surfaces). Consistent with our previous findings in studies with the same same rat model, where coconut and castor oils could not result in motivating results, we think factor or factors other than oily nature of sweet almond oil seem to play a role in the treatment of ALP toxicity. Providing the animals with a vitamin E enriched substance or role of almond oil as a mucosal stabilizer could be a possible antidote effect. In conclusion, on the basis of above study we can conclude that we can consider sweet almond oil as an effective agent in lowering mortality rate in ALP poisoning but some extended trials will be needed to confirm the efficacy. On the other hand, although a cholinesterase inhibiting effect has been proposed for ALP, other mechanisms of toxicity in this setting should be considered.
Footnotes
Acknowledgement
We wish to thank Mr. Gholamreza Bayat for his technical assistance.
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.