ASSESSMENT OF PROTECTIVE EFFECTS OF ALMOND SEED AND VITAMIN E SUPPLEMENTATION ON KIDNEY AND LIVER OF RATS EXPOSED TO PARACETAMOL TOXICITY

Discussion:-This research work assessed the effect of vitamin E and almond seeds on paracetamol induced nephrotoxicity and hepatotoxicity. Preventive (pre-treatment) and curative (post-treatment) studies were carried out. For the preventive (pre-treatment) groups, the rats were given almond extract, vitamin E and a combination of both before induction of hepatorenal toxicity on day fourteen (14) while for the curative (post-treatment) groups, the rats were first induced on day one (1) before treatment with almond extract, vitamin E and combination of both. Both preventive and curative groups had their controls and also a negative control (normal).


ISSN: 2320-5407
Int. J. Adv. Res. 8(05), 397-406 398 induced nephrotoxicity accounts for 20% of all cases of community and hospital acquired episodes of acute kidney injuries Zimmerman et al.,2009). Hepatotoxicity is a term applied to liver damage respectively due to toxins. It has become an increasingly important issue with the increase in prescription medication and herbal supplement use. Age, pre-existing disease conditions, multiple medications, exposure to more diagnostic and therapeutic procedures are also contributory factorsto high prevalence. Drug induced toxicity is the most common form of acute liver failure in the united states (Larson et al., 2005) and hence one of the reasons for withdrawal of drugs from the market. Treatment can be costly and may require multiple interventions, including hospitalization (Gandhi et al., 2000).
The liver plays a vital role in regulating various physiochemical functions of the body and is always the first target organ for the metabolism of drugs and toxic chemicals (Sahreenet al., 2015;Salamaet al., 2015). The liver has the ability to convert xenobiotics to compounds with low toxicity and also excrete them due to the high concentration of toxin-metabolizing enzymes they possess. Although, sometimes hepatic damage can beinduced due to conversion of toxic substances to active metabolites during metabolism (Cullen, 2005). Mitochondrial dysfunction has been reported as one of the major mechanisms of drug induced hepatotoxicity. Severe mitochondrial dysfunction in the liver as a result of drugs can induce hepatic necrosis, cystolytic hepatitis and eventually liver failure (Gopiet al., 2010). Damage to the liver causes accumulation of toxins in the body faster than it can process.
The kidney is also a sensitive and dynamic organ responsible for homeostasis and regulation of the extracellular environment. It is also involved in detoxification and excretion of toxic metabolites and drugs (Inui et al., 2010;Parazella and Mockel, 2010). Drugs can exert their toxic effects by inducing nephrotoxicity by one or more common pathogenic mechanisms. Most people suffer from drug induced nephrotoxicity because of the presence of dangerous factors that increases their susceptibility to the damage caused by drugs (Tionget al., 2014). Drug-induced nephrotoxicity is an important cause of renal injury (Padmini& Kumar, 2012) and has been found to cause approximately 20% of community and hospital acquired episodes of AKI Bellomo, 2006). Paracetamol (PCM), is an analgesic and antipyretic drug (Aghababian, 2010). At recommended doses, it is generally safe even when taken for a long time. However, paracetamol can be very toxic and fatal when taken at overdose (FDA, 2008). Acute overdose of paracetamol is dose-dependent and causes potentially fatal hepatic necrosis which may be associated with renal tubular necrosis (Ogunbayodeet al., 2010). At therapeutic doses, acetaminophen is metabolized via glucuronidation and sulfation reactions occurring primarily in the liver which result in the watersoluble metabolites that are excreted via the kidney. Paracetamol toxicity is dose dependent and increased overdose causes massive depletion of glutathione stores and increased production of NAPQI metabolites which are unbound and highly reactive. These electrophilic intermediates form adducts with cellular proteins, which disrupts homeostasis, activates caspases and lysosomal enzymes that initiate apoptosis (Bessems and Vermeulen, 2001;Gopiet al., 2010 Vitamin E is a group of lipid-soluble compounds which includes tocopherols and tocotrienols . It carries out its function by acting as peroxyl radical scavenger, thus preventing the propagation of free radicals in tissues (Traber and Stevens, 2011). It also facilitates membrane repair by preventing the formation of oxidized phospholipids that theoretically might interfere with the membrane fusion events. Vitamin E has been shown to protect cell membrane from acetaminophen induced toxicity by inhibiting lipid peroxidation (Herrera and Barbas, 2001;. Vitamin E also protects against some forms of xenobiotics that causes damage (Khaster, 2015).
Almonds are natural antioxidants owing to its phytochemical constituents. Some epidemiological studies have shown that the consumption of foods rich in natural antioxidants increases plasma antioxidant capacity and reduces the risk of diseases (Frisson-Norrie and Sporns, 2002;Philips et al., 2005). Several studies have revealed that antioxidant activity may be ascribed more to compounds such as flavonoids, isoflavones, flavones and so on. The almond is an effective health building food, both for the body and the mind. It is also a valuable food remedy for several common ailments. The seeds of Prunusamygdalus are found to possess various pharmacological properties, such as anti-stress (Bansal et (Spiller et al., 1998). Despite the considerable advancements in medicine, synthetic drugs still have many side effects and exacerbate the disease. It has become necessary to use natural and safe alternatives from medicinal plants to replace chemical drugs using various experimental models Rivera-Espinoza 2008, Patwardhanet al. 2004). In this context, more attention has been paid to the protective effects of natural antioxidants 399 of herbal medicine and isolated bioactive constituents, which are considered as the most effective and safe treatments for hepatotoxicity/nephrotoxicity (Grajales and Muriel, 2015).
The high incidence of liver and kidney damage as a result of drug induced toxicity and its impact on human health have motivated studies on medicinal plants which have been claimed to possess protective characteristics against drug toxicity. Presently, the use of herbal medicines for prevention and control of diseases is in the focus of attention for the physicians, pharmaceutical manufacturers and patients. The reasons for such shift toward the use of herbals include the expensive cost of conventional drugs, inefficacy of drugs and adverse drug reactions (Aghelet al., 2007) It is therefore of scientific and economic interest to find out if consumption of vitamin E and/or almond seeds would ameliorate and/or prevent hepatotoxicity caused by drugs in albino rats.

Chemicals:
All drugs and chemicals were analytical grade. Vitamin E in gelatin capsule and powdered paracetamol was purchased from standard vendors

Pilot Study:
A total of 4 adult male albino rats weighing 150 -170g, were used for the study. After 2 weeks of acclimatization, the rats were weighed and randomly put into individual cages. Two were designated PCM group and the other two, control group. All the rats were fed on standardized rat chow and clean tap water. PCM group were given single dose of paracetamol 3g/kg body weight. Blood samples were collected by cardiac puncture after 24 hours for analysis of liver variables and oxidative profile.

Study Design and Population:
A total of 70 rats were used for this study. Four (4) rats were used for pilot study; fifteen (15) rats were used for acute toxicity study while fifty-one (51) male albino rats were sampled for the main study.

Acquisition and Acclimatization of Animals:
Seventy (70) male albino rats weighing between 130 -150 g were acquired from a known animal. The rats were kept in the animal house throughout the study at an average temperature of 30 o C, relative humidity of 80% and a 12-hour light/dark cycle. The animals were put on standardized rat feed and water for 2 weeks to acclimatize. Fifty-one albino rats were randomly grouped into 9 groups with five animals each in groups A, B, C and six animals each in groups D, E, F, G, H and I. Procedures involving the care and use of the animals were done in compliance with standard guidelines for the use of animals in biomedical research.

Preparation of Paracetamol (Acetaminophen):
Powdered paracetamol was suspended in distilled water and administered orally at a dose of 3g/kg b.w. This dosage is known to cause hepato-renal toxicity in rats according to the pilot study.

Extraction of Almond Seeds:
Bligh and Dyer method for lipid extraction was used (Bligh and Dyer, 1959). Seven hundred and fifty grams (750g) of almond seeds were weighed and blended using an electric grinder. The extraction was done by adding 1.5 litre of methanol to the grinded almond seeds, stirring and allowing it to stand for 24 hours at room temperature (26 -28 o C). After 24 hours, the mixture was filtered using Whatman No. 1 filter paper. The methanol filtrate was then concentrated by drying with a rotary evaporator at 40-60 o C. The essence of drying in a rotary evaporator is to gently remove the solvent used (methanol). The percentage yield after extraction was 10 percent. The extract obtained was then further dried under the sun and stored in an air-tight plastic container in the refrigerator (4 o C) and used for the study.

Induction of Hepato-nephrotoxicity:
Having established from the pilot study that a single dose of 3g/kg b.w of paracetamol was adequate to induce liver dysfunction in the rats, nephrotoxicity was induced by weighing each animal on a weighing scale (Hana brand) and calculating the corresponding dose of paracetamol required for the induction. Powdered paracetamol was suspended in distilled water and administered orally at a dose of 3g/kg b.w. The animals were starved for 24 hours before the commencement of the experiment but had free access to drinking water.

Extract and Drug Administration:
Based on the pre-determined LD50 values of almond extract obtained by using Lorke's method, the dose 1000mg/kg b.w was used for this study. The extract was diluted in distilled water which acted as a vehicle and administered orally through gastric gavage. Vitamin E 100mg/kg b.w was administered also by gastric gavage. A tiny drop of tween 80 was added to increase Vitamin E solubility in distilled water before administration. Single dose of paracetamol 3g/kg b.w was administered orally by gastric gavage to induce nephrotoxicity and hepatotoxicity.

Calculation of Dosage:
Dose of paracetamol and Vitamin E corresponding to the average weight of the rats were calculated based on: Average weight = total weight of rats / total number of rats Experimental Design: PCM is paracetamol, Post-C is post-treatment control group, Pre-C is pre-treatment control group, vit. E is vitamin E. PCM=3g/kg, vit E=100mg/kg, Almond=1g/kg. This experimental setup lasted for a period of fourteen (14) days. On the 15 th day, all the rats were sacrificed and blood collected for biochemical analysis.

Collection of Blood Samples for Biochemical Analysis:
Blood samples were taken by cardiac puncture (after anaesthetizing the animals with diethyl ether inhalation) at the commencement (as baseline measures) of the study, before and after interventions (sacrifice of the animals). This was done on day 15. About 4ml of whole blood was collected from each animal into plain sample bottle,labeled properly for biochemical analysis (urea, creatinine, AST, ALT and ALP).

Statistical Analysis:
Data were analyzed using Graph Pad Prism 5.1. The data were presented as mean and standard deviations. Statistical comparisons were considered significant at p < 0.05.

Results:-Renal Indices and Liver Enzymes Levels of the Various Group:
Details of the renal indices and liver enzymes of various groups are shown in

Renal Indices and Liver Enzymes Levels of the Post-Treatment Groups and Control:
The details of the renal indices and liver enzymes levels of the post-treatment groups and control are shown in table 3.2.
The table depicted significant differences in urea levels amongst group A vs B, group A vs E, and group A vs F at (p = 0.013, F = 4.104). There were no significant variations among the rest of the groups.
The table also shows a significant variation in creatinine levels amongst group A vs B at (p = 0.0026, F = 5.823), but there were no significant variations among the rest groups.
There was also a significant variation in the AST level between group A vs B at (p = 0.0267, F 3.413). Although no significant differences exist among the rest of the groups.
The table also portrays significant variations in ALT levels amongst group A vs B, group A vs D, group A vs E and group B vs F at (p = 0.0002, F = 9.321).
The results for ALP shows no significant variation amongst all the groups at (p = 0.2066, F = 1.619).

Renal Indices and Liver Enzymes Levels of the Pre-treatment Groups and Control:
The details of the results of the renal indices and liver enzymes levels of the pre-treatment groups and control are shown in table 3.3.
The table depicted significant variations in the urea levels amongst group A vs C, group A vs G, group A vs H, group A vs I, group C vs G, group C vs H, group C vs I and group G vs I at (P = 0.0001, F = 36.31). The rest groups showed no significant differences amongst them.There were also significant differences observed in creatinine levels amongst group A vs C, group A vs G, group A vs H and group A vs I at (P = 0.0001, F = 14.18). There are no significant differences amongst the rest of the groups.
Results for ALT levels also shows significant differences amongst group A vs C and group A vs G at (p = 0.0308, F = 3.28). The rest groups show significant differences amongst them. Results for ALT level shows significant differences between group A vs C and group A vs G at (p = 0.009, F = 4.474). There were no significant differences amongst the rest groups.
Results for ALP levels shows no significant variations amongst all the groups at (p = 0.334, F = 1.215).

Discussion:-
This research work assessed the effect of vitamin E and almond seeds on paracetamol induced nephrotoxicity and hepatotoxicity. Preventive (pre-treatment) and curative (post-treatment) studies were carried out. For the preventive (pre-treatment) groups, the rats were given almond extract, vitamin E and a combination of both before induction of hepatorenal toxicity on day fourteen (14) while for the curative (post-treatment) groups, the rats were first induced on day one (1) before treatment with almond extract, vitamin E and combination of both. Both preventive and curative groups had their controls and also a negative control (normal).
In this study, oral administration of 3g/kg of paracetamol produced a significant(p<0.05) increase in urea levels in the paracetamol control groups (group B and group C) when compared with the value for rats that were not induced (group A). Creatinine values were also significantly increased (p<0.05) in the paracetamol induced controls (group B and group C) when compared with values for the rats that were not induced (group A). The rise in both urea and creatinine levels is suggestive of acute kidney injury caused by the oral administration of 3g/kg of paracetamol. It has been previously reported that an acute paracetamol overdose can lead to potentially lethal kidney failure in humans and experimental animals (Schismitaet al., 2015;Blakely and McDonald, 1995;Emeighet al., 1996;Eguia andMaterson, 1997). Tubular cell loss has been reported to be a characteristic feature of both acute renal failure and chronic renal disease (Ortizet al., 2000) and is observed when cell death predominates over mitosis. It has been extensively studied and well understood that Paracetamol causes liver damage by promoting hepatocyte apoptosis (Rayet al., 1993;Ray and Jena, 2000;Boulareset al., 2002).However, the mode of renal cell death during paracetamol nephrotoxicity and the mechanisms involved are obscure. Indeed, there is evidence that the molecular basis of nephrotoxicity may differ from those of hepatotoxicity, as N-acetyl-cysteine protects from the latter, but has been shown not to protect from nephrotoxicity, (Blakely and McDonald, 1995). Corina and colleagues however reported that induction of apoptosis may underlie the nephrotoxic potential of paracetamol and they identified renal endoplasmic reticulum stress as a therapeutic target in nephrotoxicity, ( Post treatment after inducing nephrotoxicity shows non-significant (P>0.05) decrease in urea levels of almond group (group D), Vitamin E (group E) and combined treatment group (group F) compared with the paracetamol group (group B). For almonds, there is no reported study of effect on diseased kidney. However, in human study conducted by Yngbar et al, it was rather reported that renal failure occurred due to excessive intake of almond, (Yngbaret. al, 2015).
Comparing the urea levels of the pre-treatment group and post treatment groups showed significant (P<0.05) variation in almond groups (P < 0.0001, T=7.55). The vitamin E and combined treatment groups showed no significant variation.

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Creatinine levels of the pre-treated groups showed a non-significant decrease in the almond group (group G) compared to paracetamol control (group C). However, vitamin E (group H) and combined treatment group (group I) showed significant (p< 0.05) decrease in creatinine levels. Creatinine levels are lowest for the combined treatment group (group I). Previous studies have also demonstrated renoprotective effect of vitamin E (Nazirogluet al., 2004; Wariboet al., 2017). Vitamin E is a powerful peroxylredical scavenger that returns to its reduced state by reacting with vitamin C (or other hydrogen donors), (Traber and Stevens, 2011). Moreover, it reduces the mitochordrial generation of hydrogen peroxide and it regulates the expression of genes implicated in inflammation and fibrosis which occurs in CKD patients (Yoshikawa et al., 1998). For the combined treatment, no known studies have been reported on almonds and vitamin E.
Creatinine levels of the post-treated groups showed a non-significant increase in almond group (group D), vitamin E (groupE) and combined treatment group (group F) when compared to the normal control (group A) whereas comparing with paracetamol control group (group B) showed non-significant decrease in creatinine levels.
Comparing the creatinine levels of the pre-treatment and post treatment groups showed significant (p<0.05) variation in almond groups (p=0.0028, T = 4.428). However, there were no significant variation in vitamin E groups and combined treatment groups.