Comparative Assessment of Zinc Sulphate and Essentiale Forte® in Managing Biochemical Alterations of Carbon Tetrachloride-Induced Hepatotoxicity on Adult Wistar Rats

Most routine tests reflect liver damage the tests of function are those which reflect synthetic capacity for instance albumin and prothrombin time, liver function tests may be grossly deranged when function is normal and may be normal when function is grossly deranged for it has enormous functional reserves, such that early liver impairment is clinically marked and the progression of the deranged liver function makes the condition life threatening [1]. Morphologically, liver responds to injurious events in 5 different ways, irrespective of the cause viz; Degeneration and intracellular accumulation, Necrosis and Apoptosis, Inflammation, Regeneration, Fibrosis [1].

CCl₄ is a colourless liquid, ether-like in odour with a density of l.6gcm-3 melting point is 22.9OC, boiling point is 76.7 OC and soluble in water at 0.08g/100ml (25 OC). It is also soluble in alcohol, ether, chloroform, benzene, naptha and carbon sulfide. The vapour pressure is 11.94kPa at 20 OC and refractive index of 1.5, it has crystal structure with tetrahedral shape. It is not flammable, its auto-ignition tempt is 982 OC and LD₅₀ is 2350mg/kg. International programme on chemical safety (IPCS) (1999) It has been reported to produce free radical which affects the cellular permeability of the liver cells leading to altered level of serum biochemistry and liver enzymes [2]. In time series, it was found to have an atmospheric life time of 85 years and liver damage inflicted by CCl₄ has lethal consequences.

For so many years, liver disease remains one of the major health concern despite discovery and development of new drugs, morbidity and mortality accompanying hepatic pathology is still clinically significant [3]. Chronic exposure of liver to CCl₄, has been researched with consequent spectrum of chronic liver disease, such as fatty liver, hepatitis, liver cirrhosis and hepatoma [3]. Liver damage can occur after 24hrs of exposure to CCl₄ and in serious cases this can result in hepatitis, ascites, hemorrhages, hepatic coma and even death. International programme on chemical safety (IPCS) [4].

Essential phospholipid has since been used in the cases and managements of chronic liver disease. It is a 300mg hard gel capsule that contains De-oiled enriched phospholipids from neutral soya beans. Its mechanism of work by phospholipids replacement and regeneration in the liver cell membranes, which have been damaged by various means especially through liver toxin. The pharmaceutical excepients are chiefly ethanol, hard fat, Hydrogenated, Castor oil, Ethyl vanillin, 4-methoxylphenylethanone, Alpha-tocopherol, Gelatin, Colouring agents, Sodium laurylsulfate, Purified water. Sonafi avens is pharmaceutical.

Zinc is known as an essential trace element necessary for protein metabolism, as well as membrane integrity and also involved in the structure and function of numerous metalloenzymes. It has important functions in skin and connective tissue, metabolism as well as in wound healing [5]. It exerts its antioxidant effects indirectly by maintaining membrane structures, involving in the structure of SOD, increasing the metallothionein concentrations and, competing with redox reactive metals, iron and cuprous for critical binding sites [6]. It is shown that hepatic and serum zinc levels of patients in liver disease decreased depending on the degree of liver damage [7].

Animal care and management

Twenty five adult Wistar rats, weighing between 150g and 170g (6-10 weeks old) obtained from Animal Holding of International Institute of Tropical Agriculture Ibadan Oyo State were used for the research. The animals were housed in plastic cages in a clean environment of 12 hours day/light cycle, at room temperature, in the animal holding of the Department of Anatomy and Cell Biology. They were fed on standard laboratory rat pellets and have free access to water. Ethical clearance for the study was obtained from Health Research Ethical Committee (HREC), Institute of Public Health (IPH) Obafemi Awolowo University(OAU) Ile-Ife. The animals were given humane care according to the guidelines of HREC, IPHOAU.

Preparation of the chemicals/drugs

2.5 litres of CCl₄ was obtained from the central research laboratory of Obafemi Awolowo University, Olive Oil, Zinc Sulphate (ZnSO₄) tablets and Essentiale Forte® capsules were of the best grade commercially available. 60ml of CCl₄ was diluted with 60ml of olive Oil in 1:1 equivalent, and this was administered at a dose of 0.7ml/kg p.o. 3 tablets of ZnSO₄ was dissolved in 60ml of water and this was administered at a dose of 7ml/kg, Essentiale Forte® capsule was prepared by dissolving a capsule in 60ml of water, and was administered at a dose of 4.5mg/kg all being freshly prepared on each day of administration.

Animal treatment

The rats were divided into five groups A, B, C, D and E of five rats each (n=5). Group (A) normal control, received oral administration of olive oil only. Group B negative control received daily administration of CCl4 (0.7ml/kg p.o) for 1 week in 1:1 dilution with olive oil without treatment. Groups C test group I received Essentiale Forte® 4.5mg/kg/day for four weeks after the administration of CCl₄. Group D test group II received Zinc Sulphate® (7mg/kg/day p.o) daily for four weeks after the administration of CCl₄. Group E test group III received Zinc Sulphate® 7mg/kg/day for four weeks and Essentiale Forte (4.5mg/kg/day p.o) for a period of 4 weeks concurrently, after CCl4 administration. All administrations were via oral for four weeks (Essentiale Forte® and Zinc Sulphate®) while CCl₄ was for one week.

Animal sacrifice and sample collection

At the end of the experimental procedure the animals were sacrificed after one week of recovery period. Animals were euthanized by ether anaesthesia a midline incision was made along the anterior abdominal wall. The blood was taken by cardiac puncture, the abdomen of the sacrificed animal will be cut open quickly and the liver per fused with isotonic saline, excised, blotted dry, weighed, and divided into samples. Liver tissues were excised from sacrificed animals, weighed, and thin liver slices were cut, fixed and were sequentially embedded in paraffin wax blocks. Five μ thick sections were cut, and stained with Hematoxylin-Eosin (HE) and Masson’s Trichrome for conventional morphological evaluation, then examined under light microscope (Olympus). The degree of hepatic necrosis and fibrosis were determined by a semi-quantitative method [8]. Some portions of the liver tissues were homogenized for biochemical assay (ALT, AST, ALP) and the rest fixed in 10% formal saline for subsequent routine histological procedures.

Serum and homogenate Alanine, Aspartate Aminotransferases and Alkaline Phosphatase (ALT, AST and ALP) Whole blood was centrifuged at 4700rpm for 10min at 4 OC and ALT, AST and ALP were determined spectrophotometrically with an automatic analyzer (Cobas Mira; Roche, Rotkreuz, Switzerland) using commercially available kits (Randox Diagnostics). Their activities were expressed as an international unit (IU/L). ALT, AST and ALP activities in liver homogenates were also determined using a quantitative, colorimetric end-point Randox assay kit (Procedure No. 104, Sigma Diagnostics, St. Louis, MO) that used α-ketoglutaric acid as the substrate and that detected production of pyruvic acid. Results were expressed as international units (IU).

One way ANOVA was used to analyze the data, followed by Student Newman-Keuls (SNK) test for multiple comparisons. Graph Pad Prism 5 (Version 5.03 Graph Pad Inc.) was the statistical package used for data analysis. Significant difference was set at p<0.05.

Hepatic enzyme levels in the Control and test groups (Table 1 and Figure (1-5)).

Figure 1 The graph showing the ALP levels of rats across the group after treatment.

Results presented as mean±SEM(n=5); α: Significantly different from normal control at p<0.05; β: Significantly different from toxic control at p<0.05; δ: Significantly different from C,D and E at p<0.05

Figure 2 The graph showing AST levels of the rats across the group after treatment.

Results presented as mean±SEM(n=5); α: Significantly different from normal control at p<0.05; β: Significantly different from toxic control at p<0.05; δ: Significantly different from C,D and E at p<0.05

Table 1 Hepatic enzyme levels in the control and test groups.

Results presented as mean±SEM(n=5); α: Significantly different from normal control at p<0.05; β: Significantly different from toxic control at p<0.05; δ: Significantly different from C,D and E at p<0.05; EPL: Essentiale forte®; CCl₄: Carbon tetrachloride; ZnSO₄: Zinc Sulphate

Figure 3 The graph showing ALT levels of the rats across the group after treatment.

Results presented as mean±SEM(n=5); α: Significantly different from normal control at p<0.05; β: Significantly different from toxic control at p<0.05; δ: Significantly different from C,D and E at p<0.05

Figure 4 The graph showing the packed cell volume of the rats across the group after treatment.

Results presented as mean±SEM(n=5); α: Significantly different from normal control at p<0.05; β: Significantly different from toxic control at p<0.05; δ: Significantly different from C,D and E at p<0.05

Figure 5 The graph showing the mean weight change of the rats across the group after treatment.

Results presented as mean±SEM(n=5); α: Significantly different from normal control at p<0.05; β: Significantly different from toxic control at p<0.05; δ: Significantly different from C,D and E at p<0.05

Serum hepatobiliary enzymes, are present in high concentrations in the liver in stressful conditions. When there is hepatocyte necrosis or membrane damage, these enzymes are released into the circulation, as indicated by elevated serum enzyme levels. Zn was not able to decrease the levels of AST, ALT and ALP as compared to the standard drug essentiale forte but when combined, the enzyme levels were markedly reduced. Significant reduction in the levels of these enzymes in the double treated groups indicated that Zn in combination with Essentiale forte was able to offer better protection to the liver against CCl₄ induced hepatotoxicity.

Zn treatment was able to ameliorate CCl₄- induced hepatocellular damage as evidenced by reversal of increased serum transaminase (AST and ALT) levels subsequent to exposure. The enzyme levels in group B was found to be markedly elevated to almost three folds however, this enzyme level was quickly reverted to normal after treatments. Moreover, the finds of this research was contrary to the result of the work by Alumot et al. [9] which reported no significant effects on serum enzyme levels or hepatic fat content of rats exposed to doses of CCl4. CCl4 toxicity was also found to have led to relative reduction in the haematocrit in group B unlike the other groups which is in keeping with the work done by Guild et al.[10] & Stewart et al. [11] which found that focal hemorrhagic lesions in the gastrointestinal tract and mild anemia were observed in humans who have ingested Carbon tetrachloride but this is likely due to decreased hepatic synthesis and or secretion of clotting factors. However it is contrary to the finds of Hayes et al. [12]. Oral exposure of mice to carbon tetrachloride did not result in any consistently significant hematological change. Moreover, the work done by Bruckner et al. [13] stated that; severity of hepatic lesions as evidenced by centrilobular fatty vacuolization sometimes with single cell necrosis was correlated with the level of increase in serum enzyme levels [14-64].

Combined administration of Zn and Essentiale forte may be considered as more potentially and synergistically therapeutic through deterrence of enzyme leakage mechanism and thereby inhibiting liver toxicity induced by CCl₄. Zn and or Essentiale forte® has hepatoprotective activity against carbon tetrachloride-induced liver damage, this activity could be due to the presence of flavinoids in EPL and membrane stabilization ability in both, thereby preventing cellular leakage. However, further studies are needed to expound the possible prophylactic and or protective effect of the zinc sulphate and essential forte combination in managing chronic liver disease clinically.

Further studies are recommended to give details about the histomorphometric correlation of hepatocytes and kupffer cells in liver of rats treated with CCl₄ vis a vis hepatic damage, possible relationship between the zinc level and the degree of liver damage via Zinc bioassay and possible prophylactic effect of the Zinc Sulphate and Essential forte® combination in managing chemicalinduced injury in liver disease.

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