Introduction

Malaria is one of the most common parasitic diseases causing morbidity or mortality in the tropic. It is caused by parasites that belong to genus plasmodium and it spends part of its life cycle in the red blood cells. Digestion of the host haemoglobin during maturation of the parasites within the red cell followed by the lyses of such red cells result in the release of reactive oxygen species (ROS) and iron from the haem moiety of the haemoglobin increasing oxidant stress with the cell and thus compromising the integrity of the cell (Huy et al, 2003, George and Nmoka, 2003). Also malaria infection would activate phagocytes to produce H₂O₂ and O₂ radicals as one of the mechanisms to destroy invading bacteria (Halliwel, 1997). All these factors make the red cells of individuals infected with malaria parasites more susceptible to oxidative stress, lysis, and consequently causing anaemia in such individuals (Mohan et al, 1994).

In addition, halofantrine, a lipophilic phenanthrene methanol belonging to aryl-amino alcohol and used in the treatment of acute uncomplicated multi-drug resistant malaria (Philips- Howard and wood, 1996). It has been found to act directly or indirectly and alters antioxidant status. This reduced antioxidant status makes certain organs more susceptible to oxidative stress. Several studies have shown that halofantrine causes increased lipid peroxidation and decreased enzymic and non-enzymic antioxidants (Gibson and Skett, 1992, Murphy et al, 1983).

The potential of dietary antioxidant to detoxify the H₂O₂ and O₂ radicals produced during malaria infection and more so during antimalaria treatment has received increasing attention in recent years (Murugavel and Pari, 2004; Huy et al, 2003; Mohan et al 1994). The nutrients that have shown promise as protective antioxidants are lipid-soluble antioxidants such as vitamins E, A, α- carotene, β-carotene and lycopene in humans because of their association with membrane lipids (Hortons and Fairhurst, 1987; Metzger et al, 2001). Vitamin E acts as the most potent lipid soluble antioxidant. Vitamin C another nutrient, serves directly as an antioxidant by scavenging aqueous peroxyl radicals and indirectly by regenerating reduced Vitamin E (Frel, 1991).

Pari and Amali (2005) have reported the protective role of tetrahydrocurcumin (HTC) an active principle of turmeric on chloroquine-induced hepatoxicity in rats. Also George and Nmoka (2003) have reported the effects of vitamin C and E supplementation in the treatment of malarial infection. However, there is paucity of information regarding the ability of these vitamins (C and E), whether ingested alone or in combination, to protect against halofantrine-induced oxidative stress. It is therefore, the purpose of this work to examine the protective role of vitamins C and E on halofantrine-induced hepatotoxicity in rats.

Materials And Methods

Animals

Drugs

experimental designs

Blood Sample Collection

Biochemical Analysis

Serum Liver Function Markers

Lipid Peroxidation Marker

Antioxidant Status

Statistical Analysis

Result

The body weight changes and the relative liver weights in both normal and experimental animals are shown in table 1. There was a slight gain in body weight of animals in group I, III, IV and V whereas the group II showed a slight loss of weight. There was a significant increase in the relative weight of the liver in group II when compared with the group I (control) which significantly reversed to near normal in group III, IV and V (Table I).

Figure 1

Table 1: The mean values of body weight change and mean relative weight of the liver in normal and experimental animals.

Table 2 showed the changes in the concentration of serum total bilirubin and in the activities of AST, ALT, and ALP in both normal and experimental animals. The significantly increase activities of AST, ALT, ALP and the concentration of bilirubin due to halofantrine treatment (group II) were significantly decreased (P<0.05) on treatment with vitamin C (group III), vitamin E (Group IV) and combined treatment of vitamin C and E (Group V).

Figure 2

Table 2: Mean values of activities of serum AST, ALT, ALP and levels of bilirubin in normal and experimental rats.

The levels of plasma and liver lipid peroxidation product (malondialdehyde, MDA) and antioxidants such as vitamin C, E, SOD and CAT in control and experimental animals are shown in table III. The level of MDA was significantly increased (P<0.05) in rats treated with halofantrine when compared with normal rats. Combined treatment of halofantrine with vitamin C, vitamin E alone or combined vitamin C and E significantly decreased the level of lipid peroxidation product.. Combined treatment with vitamin C and E was highly effective when compared with vitamin C or E alone. The levels of antioxidant (vitamin C, E, SOD and CAT) were significantly depleted in rats treated with halofantrine. Treatment with vitamin C, E, (C and E) considerably increased the levels of the antioxidants (vitamin C, E, SOD, CAT) in halofantrine treated rats to near normal. The combined vitamin C and E showed a better effect than vitamin C or E alone.

Figure 3

Table 3: Mean values of plasma and liver MDA, Vitamin C, E, SOD, CAT in normal and experimental rats.

Discussion

The observed increase in the relative weight of the liver in the halofantrine treated group indicates that the drug might have toxic effect on this organ. It has been reported that increase or decrease in either absolute or relative weight of an organ after administering of a chemical or drugs is an indication of the toxic effect of that chemical (Simons et al, 1995; Orisakwe et al, 2003). Upon administration of vitamin C, E, or (C and E) to halofantrine treated rats, the relative weights of the livers reversed to near normal.

The activities of AST, ALT, ALP and the level of bilirubin increased significantly in halofantrine treated group. Liver enzymes are usually raised in acute hepatotoxicity, but tend to decrease with prolonged intoxication due to damage to the liver (Cornelius, 1979). This observation was confirmed by Obi et al, (2004). Other antimalarials such as chloroquine (Pari and Amali, 2005), amodiaquine (Farombi et al, 2000) and quinine (Debra and Megan, 1999) were also reported to induce hepatic damage. Vitamin C, E, (C and E) treatment significantly decreased the activities AST, ALT, ALP and the level of bilirubin in serum of halofantrine treated rats. Thus suggesting that they offer protection by preserving the structural integrity of hepatocellular membrane against halofantrine. This finding could be correlated with previous works, which reported that treatment with tetrahydrocurcumin (an antioxidative substance, which is derived from curcumin, the component of turmeric) significantly reduced the activities of serum hepatoxicity markers in chloroquine-induced hepatoxicity (Pari and Amali, 2005) and erythromycin-induced hepatoxicity (Pari, 2004).

The observed significant high levels of plasma and liver MDA concentrations (end product of lipid peroxidation) and decreased level of some antioxidants like vitamin C, E, SOD and CAT in rats treated with halofantrine reflected the lipid peroxidation as a consequence of oxidative stress. The measurement of lipid peroxidation is a convenient method to monitor oxidative damage (Viani et al, 1991). In addition, non-enzymic antioxidants such as vitamin C and E play excellent role in protecting the cells from oxidative damage (Nwanjo and Ojiako, 2005). It has been shown that vitamin E (α-tocopherol) scavenges peroxyl radicals quicker than vitamin C (ascorbic acid); the α-tocopheroxyl radical derived from tocopherol is reduced back to regenerate α-tocopherol by ascorbic acid (Packer, 1992; Nwanjo and Ojiako, 2005). The enzymic antioxidant defence systems (SOD and CAT) are the natural protector against lipid peroxidation. SOD and CAT are important scavengers of superoxide ion and hydrogen peroxide. These enzymes prevent generation of hydroxyl radical and protect the cellular constituents from oxidative damage (Ugochukwu et al, 2003). This observed decrease in antioxidants also suggested increased lipid peroxidation.

The decreased level of plasma and liver MDA and increase antioxidants (vitamin C, E, SOD and CAT) in Vitamin C, E, and (C and E) supplemented rats may result from the scavenging of the free radicals generated by halofantrine induced peroxidation by these antioxidant vitamins thereby reducing the utilization of these antioxidant enzymes to reduce the halofantrine induced oxidative stress. It also increases the antioxidant vitamins in these rats.

A number of conclusions are clearly evident from this work. The first is that vitamin C and E exert significant protection against halofantrine-induced toxicity by its ability to ameliorate the lipid peroxidation through their free radicals scavenging ability, which enhanced the levels of antioxidant defense system. This study also showed that combined supplementation of vitamin C and E has greater effect than when taken separately, in halofantrine-induced lipid peroxidation. Such a beneficial combination may therefore be recommended to patients taking antimalarial drugs like halofantrine or other drugs that generate oxidative stress.

Correspondence to

Harrison Ugo Nwanjo,Ph.D, Department of Medical Lab. Science, Imo State University, Owerri, Nigeria. E-mail – harrisonnwanjo@yahoo.co.uk GSM-08033525389