Effect of Methanol Leaf Extract of Vitex Doniana on Cadmium Chloride-Induced Toxicity in Kidney and Liver Tissues of Male Wistar Rats

The aim of this study was to investigate the effects of methanol leaf extract of Vitex doniana (MEVd) on Cadmium chloride induced toxicity in the liver and kidney of male Wistar rats was investigated. Twenty five male Wistar rats weighing 171.87 ± 7.86g were acclimatized for two weeks and subsequently randomized into five groups: Group 1 rats were administered daily 1 ml distilled water (control group); group 2 were administered Cadmium chloride (10 mg/kg b.w.) only; groups 3 and 4 were administered MEVd (200 and 400 mg/kg b.w. respectively) six hours prior administration of Cd (10 mg Cd/kg b.w.) and group 5 rats were administered MEVd (400 mg/kg/b.w.) only. The experiment lasted six weeks and all administration was carried out orally. The final body weights of the rats were recorded at the end of the experiment and afterwards the rats were sacrificed, blood was collected and the serum was subsequently prepared. The kidneys and liver were excised, weighed and then stored in 10% formalin for histological examination. The serum was used for the determination of urea, creatinine, Na+, K+, and Clconcentrations, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and total bilirubin (T-Bil) concentration. The results showed that exposure of the rats to Cd significantly (p < 0.05) increased kidney weight, serum urea, creatinine, Na+, K+, Cland total bilirubin concentrations as well as serum ALT and AST activities. Cd-induced toxicity also caused severe necrosis of the tubular epithelial cells in the nephrons and degeneration of the hepatocytes. Although both doses of the plant extract significantly ameliorated these changes, the amelioration at 200 mg/kg body weight of the extract compared well with the control in most of the parameters investigated. In conclusion, the oral administration of methanol leaf extract of Vitex doniana ameliorated the Cd-induced toxicity related biochemical parameters and histological changes in the rats. Keyword: Cadmium chloride, Toxicity, Liver, Kidney, Vitex doniana, Ameliorate


INTRODUCTION
Cadmium (Cd) is one of the most notorious toxic heavy metals. It is one of the major toxic environmental and industrial pollutants, obtained through consumption of Cd contaminated foods, drinking water, inhaled air, cigarette or from ingestion of polluted soil and dust (Jarup et al., 2000). Cd is generally used as components of pigments, plastic stabilizers, alloys, Nickel-Cd batteries, and in welding and electroplating in industries. It is also present in tobacco grown on contaminated soil (Ohta et al. 2000;Washington et al., 2006). Cd is spread from its natural sources into the atmosphere through volcanic activity, forest fires, and wind-blown soil particles. Other sources of Cd include phosphate fertilizers, which may possess high levels of Cd based on the origin of the rock, and also the use of contaminated sewage sludge as soil additive (Waisberg et al., 2003). Being that polyvinyl chloride (PVC) contains cadmium, toys made of PVC are potentially toxic to children. Owing to its long biological half-life of 15-30 years, the rate of disintegration of cadmium is very low; and as a result, cadmium accumulates in the blood, kidneys, liver and reproductive organs, such as placenta, testis as well as the ovaries, making it a hazardous toxic metal (Navaneethan and Rasool, 2014). However, the kidney is the primary target organ for chronic lowlevel exposure to Cd and is reflected in a number of International Journal of Trend in Scientific Research and Development (IJTSRD) ISSN: 2456-6470 @ IJTSRD | Available Online @ www.ijtsrd.com | Volume -2 | Issue -6 | Sep-Oct 2018 Page: 1307 renal function parameters including tubular necrosis (Satarug et al., 2010). This may eventually result to end-stage renal failure, abnormal blood pressure, diabetic manifestations and osteoporosis (Satarug and Moore, 2004).
In recent time, there is an increasing interest in herbal formulations because of their potent antioxidant properties. Higher intake of vegetables, fruits and whole grains that are rich in antioxidants have been associated to reduce risk of several diseases, including cancer, cardiac diseases, chronic inflammation and many degenerative diseases. Vitex doniana (V. doniana), a member of the Verbanaceae family is a perenial shrub with wide distribution in tropical West Africa such as Nigeria, and extends to some East African countries and in the savannah and high rainfall areas (Atawodi et al., 2003). V. doniana leaves are consumed in Nigeria as vegetables which are valued sources of protective foods and it is of great benefits for the maintenance of good health and disease prevention (Nnamani et al., 2007). In ethnomedicine, V. doniana leaf is utilized in the management and treatment of many diseases. It is employed in the treatment of stomach and rheumatic pains, inflammatory disorders, diarrhoea and dysentery (Iwueke et al., 2006). According to Iwu (1993), the leaves and roots of V. doniana are also utilized in the treatment of nausea, colic and epilepsy. The plant has been utilized as medication for liver disease, anodyne, stiffness, leprosy, backache, hemiplegia, conjunctivitis, rash, measles, rachitis and febrifuge. It also serve as tonic galactagogue to aid milk production in nursing mothers, sedative, digestive regulator and treatment of eye and kidney problems, and as supplement for lack of vitamin A and B (Burkill, 2000

Animals
Adult male wistar albino rats of body weight ranging from 171 ± 7.86g were obtained from the animal breeding and care facility of Federal University Lokoja, Nigeria. The animals were kept in standard rat cages at room temperature (25 ± 2 o C) with a normal 12-hour light/dark cycle and received standard commercial pelleted rat chow and water ad libitum. The rats were housed in the animal house facility of Department of Biochemistry, Kogi State University, Nigeria. Handling and treatments of rats was in conformity with standard guideline for laboratory animal care and use. The rats were allowed to acclamatiize for a period of 14 days.

Treatment and grouping of experimental animals
Cadmium was dissolved in distilled water and orally given to the male wistar albino rats at a dose of 10 mg/kg body weight (b.wt) (Al-Hashem et al., 2009) using a feeding needle. The Vitex doniana extract (MEVd) was dissolved in distilled water. Cadmium was administered in groups II, III and IV.
Twenty five male Wistar rats were randomly divided into five groups of five rats each. All treatments were done orally daily for 6 weeks. The time difference between MEVd and cadmium administration was 6 hours daily. The different dosage of MEVd adopted was to determine the more effective dose.
Group I served as control and received I ml distilled water. Group II received only Cadmium (10 mg/kg b.wt). Group III rats were treated with MEVd (200 mg/kg b.wt and Cadmium (10 mg/kg b.wt) at 6 hours interval daily. Group IV rats were treated with MEVd (400 mg/kg b.wt and Cadmium (10 mg/kg b.wt) at 6 hours interval daily. Group V rats received only MEVd (400 mg/kg b.wt) daily.

Animal sacrifice and collection of tissues
After 6-weeks of treatment, the rats were deprived of food overnight and the following day were anesthetized and sacrificed by jugular puncture. Immediately after the blood was collected, the liver and kidneys were quickly excised, trimmed of connective tissues, rinsed with saline to eliminate blood contamination, dried with blotting paper, weighed (so as to calculate the relative weight) and stored in 10% formalin for histological investigation.

Preparation of serum
The blood samples collected into plain sample tubes were allowed to clot and the serum separated by centrifugation at 1000 g for 15 minutes using a centrifuge (Surgifriend Medicals SM800B, England). The clear supernatant, the serum, was used for the assay of biochemical parameters.

Determination of biochemical parameters
The Serum creatinine concentration was determined according to the colourimetric method of Bartels and Bohmer (1973). The serum urea levels was determined by the method Berthelot's reaction (Fawcett and Scout, 1960). Sodium ion in serum was determined using the method of Trinder (1951) and Maruna (1958). Serum Potassium ion concentration was determined using the method of Terri and Sesin, (1958). The concentration of serum Chloride ion was determined using the method of Skeggs and Hochstrasser (1964). The determination of serum ALT and AST activities was carried out as outlined in Randox kits. The serum Total bilirubin concentration was determined according to the method described by Sherlock (1951) as outlined in Randox Kit.

Histological examination
The histopathology of the tissues of the kidney and liver of the rats was done using the method of Drury and Wallington (1980).

Statistical analysis
All data were expressed as mean ± SD (n=5). The statistical significance was evaluated by one-way analysis of variance (ANOVA) and individual comparisons were obtained by Duncan's multiple range test (DMRT) using SPSS (V. 20.0, Chicago, USA). Values were considered statistically significant when p∠0.05.

Percentage yield of extract
The percentage (%) yield of the methanol leaf extract of Vitex doniana was 8.25%.

Effect of MEVd on body weight of Cadmium administered rats
Administration of Cadmium chloride significantly (p < 0.05) retarded the growth (body weight gain) and increased the kidney weight of the experimental animals when compared with the distilled water treated control animals ( Figure 1). However, administration of all the doses of the extract of V. doniana significantly (p < 0.05) increased the body weight and decreased the kidney weight of the cadmium chloride administered animals towards the control value. It is worthy of note that there was a significant difference (p < 0.05) in the body weights of the apparently normal animals that were administered only the higher dose (400 mg/kg body weight) of the plant extract when compared with the distilled water administered animals (control).

Effect of MEVd on kidney function parameters of Cadmium administered rats
Administration of Cadmium chloride significantly (p < 0.05) increased the urea, creatinine, K + , Na + and Clconcentrations in the serum of the animals when compared with the distilled water treated control animals ( Table 2). The administration of all the doses of the extract significantly (p < 0.05) ameliorated and in some cases restored the trend of these Cadmium induced alterations in the biochemical parameters towards the control values (Table 2). Effect of MEVd on liver function parameters of Cadmium administered rats Furthermore, the same Cadmium administration significantly (p < 0.05) increased the activities of AST and ALT, and total bilirubin concentration in the serum of the animals. The administration of all the doses of the extract significantly (p < 0.05) ameliorated and in some cases restored the trend of these cadmium induced alterations in the biochemical parameters towards the control values (Table 3).     Histological examination of the kidney of rats administered the higher dose of leaf extract of Vitex doniana (400 mg/kg body weight) for six weeks (45 days) period showed that the plant extract had no damaging effect on the kidney.

DISCUSSION
Numerous animal models have been developed in the past few decades for studying heavy metal induced toxicity on which the efficacy of chemical compounds including phyto-chemicals and its products as ameliorative agent(s) can be evaluated.
Cadmium is a heavy metal known for its notorious role in environmental and industrial pollution which result in deleterious effects on humans and animals. Chronic exposure to Cadmium is known to cause damage to several systems and soft tissues including the kidney and liver. However the kidney is the primary target of Cadmium intoxication (Satarug et al., 2003). Therefore, the need to document agents/compounds including medicinal plants that mitigate or ameliorate this cadmium-induced toxicity cannot be overemphasized.
Six-weeks following cadmium administration, the growth (increase in body weight) of the animals was significantly retarded. Weight loss of cadmium administered rats have been linked to serum proteins reduction and severe diarrhea. It has been reported that cadmium inhibits growth (El-Demerdash et al, 2004). The increase in the kidney weight of rats administered cadmium only was caused by the retarded growth and maybe hypertrophy elicited by the renal toxicity due to cadmium exposure and cadmium accumulation in the kidney (Babaknejad et al, 2015). Exposure to heavy metal such as cadmium is damaging to glucocorticoids. The glucocorticoid hormones are pivotal in glucose regulation and also in carbohydrate, lipid and protein metabolism. Glucocorticoid dysfunction is associated with weight gain or loss (Ojo et al, 2014) Serum urea and creatinine concentrations are employed for estimating renal glomerular function.
According to the report of Chawla (2003), elevated levels of serum urea and creatinine are associated with kidney disease. Urea is the major nitrogenous end product of protein catabolism, and the major constituent of the total urinary nitrogen excretion. For this reason serum urea and creatinine levels were assayed. In the present study, the elevated levels of urea and creatinine by the Cadmium suggest dysfunction in the kidney by the toxic metal, also similar elevations in the serum electrolytes (Na + , K + and Cl -) indicates adverse effects on the tubular reabsorption of the electrolytes. These effects may be associated with the changes in the threshold of tubular reabsorption, renal blood flow, and glomerular filtration rate (Ojo et al., 2014). It is reported that increased serum urea concentration is an indication of reduced glomerular filtration rate in rats exposed to cadmium (Satarug and Moore, 2004). In addition, it is also proposed that Cadmium exert a direct toxicity on the glomeruli, thereby causing a decreased clearance of urea and creatinine (Noonan et al., 2002). In this study, the methanol leaf extract of V. doniana had an ameliorating effect against cadmium-induced toxicity in kidney, as established by the significant decrease in urea and creatinine levels in the Cadmium-exposed rats co-treated with V. doniana compared with cadmium-alone group. The reabsorption of these biomolecules and electrolytes were facilitated by all the doses of the extract.
Increased activities of serum AST and ALT are considered to be proven indicators of hepatocellar damage. Given the antioxidant/oxidant ratio in liver cells, the levels of the hepatic enzymes, AST and ALT activities may reflect liver tissue damage maybe by a distorted cell membrane permeability which results in the leakage of the enzymes from hepatic tissue to the serum (Adefegha et al., 2015). Findings from this study revealed that there was a serious necrotic hepatic damage which significantly increased the activities of AST and ALT in the serum of the cadmium intoxicated rats when compared with the normal rats administered distilled water (control group). Since the liver is the major organ responsible for the detoxification and transformation of most xenobiotics, its regulating mechanism is compromised due to accumulation of toxicants thereby leading to the tissue damage (Camargo and Martinez, 2006). The ameliorative effect of V. doniana on cadmiuminduced hepatotoxicity may be due to the antioxidant and inti-inflammatory activities of V. doniana as reported by Yakubu et al (2013) and Iwueke et al (2006) respectively. The serum total bilirubin is also one of the major indices used to assess liver function as well as disease progression (Uthandi and Ramasamy, 2011). In this study, the elevated serum total bilirubin of cadmium administered rats was completely reversed by the oral administration of the lower dose of theleaf extract. In all, the methanol leaf extract of Vitex doniana ameliorated the cadmium chloride-induced toxicity in the liver and kidney of male Wistar rats. This was evident in the significant reduction in all the elevated biochecal parameters due to cadmium toxicity and the obvious restoration of the architecture of the liver and kidney of the rats as shown in the histopathology results.