Aflatoxins especially AFB₁ are among the most common mycotoxins to which humans are exposed. Epidemiological and experimental studies have shown that aflatoxins are hepatotoxic, hepatocarcinogenic, mutagenic and teratogenic (Groopman et al., 1996; Mayura et al, 1998; Abdel-Wahhab et al, 1998, 1999; Abdel-Wahhab and Aly, 2003, 2005). Aflatoxins cause oxidative stress by increasing lipid peroxidation and decreasing enzymatic and non-enzymatic antioxidants in aflatoxin-treated animals (Abdel-Wahhab and Aly, 2003, 2005; El-Gibaly et al., 2003). Oxidative damage in the cell or tissue occurs when the concentration of reactive oxygen species (superoxide radical, hydroxyl radical and hydrogen peroxide) generated exceeds the antioxidant capability of the cell (Sies, 1991) or when the antioxidant capacity of the cell decreases. Levels of nonenzymatic antioxidants (glutathione, ascorbic acid) and enzymatic antioxidants (glutathione peroxidase, catalase and superoxide dismu tase) are the major determinants of the antioxidant defense mechanism of the cell.

Table 2 indicated that treatment with aflatoxin resulted in a significant increase in TNF- a, CEA and NO whereas these parameters were in the normal levels of the control in the animals treated with Zizyphus extract. The combined treatment resulted in a significant improvement in these parameters although it was still differing significantly from the controls. The effects of different treatment on the oxidant-antioxidant status of liver are depicted in table 3. Aflatoxin induced an oxidative stress as indicated by the significant increase in LPO level accompanied with the significant decrease in GPX and SOD. No significant difference was observed in the animal treated with the extract alone. Treatment with extract plus aflatoxin were comparable to the controls regarding GPX and SOD whereas, a significant improvement was found in LPO level although it still differs than the controls.

The histological examination of the liver of the control animals or those treated with Zizyphus extract alone showed normal central vein and hepatic cords separated with blood sinusoids (Figure 1A). Whereas the livers of animals treated with aflatoxin alone showed congested central vein, damaged bile ducts and massive vacuolar degeneration with abnormal nuclei shrunken (S) and pyknotic (P) (Figure 1B). The livers of animals treated with aflatoxin plus Zizyphus extract showed more or less normal hepatocytes compared to the controls (Figure 1C).

The histological studies of the control kidney (Figure 2A) showed a normal structure of proximal convoluted tubules and malpighian capsules in kidney tissue of control or Zizyphus extract treated rats. Treatment with aflatoxin alone resulted in the presence of hylin casts in proximal and distal tubules, lumen and cellular debris and intertubular mononuclear cellular infiltration (Figure 2B). The kidney of animals received the combined treatment of aflatoxin plus Zizyphus extract showed normal tubules and glomeruli. The nuclei of the epithelial cells lining tubules revealed different degrees of mitotic division (arrow) (Figure 2C).

The histological examination of testes of the control rats or those treated with Zizyphus extract alone showed normal structure of spermatocytes and spermatids (Fig. 3A), whereas the testes of animals treated with aflatoxin alone showed sever damage in all stages of spermatogenesis, intermixed with remnants of mitotic figure and spermatic tails (Fig. 3B). The testes of animals treated with the Zizyphus extract plus aflatoxin showed marked recovery in most of the seminiferous tubules, spermatogenic cells and spermatids (Fig. 3C).

The optical density of total protein contents were performed using image analysis system and revealed the histochemical investigation of protein reaction in liver, kidney and testes of rats within different treatment groups showed a significant increase or decrease in total protein content (Table 4)

The hepatic antioxidants represent the major defense against toxic liver injury, and they act as anti-apoptosis. In the current study, the levels of oxidative stress indices, LP and NO, significantly increased while the levels of antioxidants GPX and SOD in aflatoxin-treated group were significantly decreased comparing to their levels in controls. These results were in agreement with those reported by (Meki et al., 2004; Abdel-Wahhab and Aly, 2005).

The increased level of LPO with the decreased level of GPx and SOD indicated that LPO is one of the most pronounced factors in aflatoxin-toxicity and carcinogenicity (Rastogi et al., 2001). Moreover, Abdel-Wahhab et al. (2005) stated that both GPx and SOD are considered to be enzymatic free-radical scavengers in cells. Thus, the decrease of both enzymes leading to an indirect increase in oxidative DNA damage and suggesting that SOD plays a role in the suppression of oxygen free-radical formation and the decrease of NO generation.

The histological results reported in the current study confirmed the biochemical results and indicated that aflatoxin induced severe histological changes in both the hepatic, renal and testicular tissues. The histological changes observed in the liver and kidneys induced by aflatoxin have been documented previously (Mayura, et al., 1998; Abdel-Wahhab et al., 1998). We also observed congested central vein, damaged bile ducts and massive vacuolar degeneration with abnormal nuclei shrunken and pyknotic. Although the principal target organ for aflatoxin is the liver, necrosis and hemorrhage may also occur in other organs (i.e., kidney, heart, spleen, and pancreas) depending on factors such as animal species, dose, route, and treatment protocol (Newberne and Rogers, 1981). Few reports have demonstrated renal damage in rats (Mayura et al., 1998; Abdel-Wahhab, et al., 1998). Arora et al. (1978) reported that the kidneys also excreted AFB₁ and that the renal medulla was quite sensitive to this mycotoxin. In our studies, the kidney from rats fed aflatoxin-contaminated diet showed pathological changed typical to those reported in the previous reports and confirmed the changes of the biochemical parameters of kidney functions reported herein. The effects of aflatoxin on testis have been studied previously (Hinshelwood, et al., 2003, 2002; Agnes and Akbarsha, 2003; Verma and Nair, 2002). Ortatatli et al. (2002) reported degeneration and desquamation in the epithelium and decrease in the size and thickness of the germinative layer of the seminiferous tubules, and lowered plasma testosterone levels in adult roosters. Although androgen concentrations were not determined in AF-treated rats in the present study, aflatoxin treatment is known to result an impairment of Leydig cell function and a hypoandrogen status in rats (Egbunike, 1982; Agnes and Akbarsha, 2001)). In the testis of AF-treated rats, pathological changes were found in the Leydig cells (data not shown) and thus it may be inferred that hypoandrogen status is a probable mechanism of action of AF in bringing about the development of different pathological changes. However, a direct effect of AFB₁ or its metabolites on the epididymal epithelium cannot be ruled out.

Low cholesterol level in the animals treated with extracts alone or in combination with aflatoxin suggest that these extracts may have antiatherosclerotic properties and may protect against the development of coronary diseases (Morcos, 1997). It is well documented that Zizyphus spina christi extract is enriched in flavonoid compounds. These flavonoids compounds were found to have the ability to reduce the production of reactive oxygen species (ROS), the inhibition of protein and DNA synthesis and the apoptosis caused by aflatoxin and showed good scavenging power, in accordance with the observed inhibition of NO production (Guerra e al., 2005) and suggesting that the extract attenuated the aflatoxin-mediated decrease in the activities of GPX and SOD.

These compounds also are proton donors which act as inhibitors for the radical chain reaction (Lean and Mohamed, 1999). Abobaker et al., (1994) and Abdel-Wahhab and Aly, (2005) reported that phenolic compounds have a role in the activation and detoxification processes and hence in modulating carcinogenicity of AFB₁. According to Galvano et al., (2001), phenolic compounds at the 0.5% level caused a marked decrease in the ability of liver microsomes to metabolise AFB₁ thus preventing its activation toward epoxides and DNA adduct formation. Moreover, it was also found to induce cytosolic glutathione S-transferase activity, in turn increasing the formation of AFB₁-glutathione conjugates. Natural phenolics have the ability to reduce the enzyme activity consequent to AFB₁ treatment (Mistry et al., 1997; Abdel-Wahhab and Aly, 2005)), accordingly, the suppression of protein kinase C activity by phenolic compounds could by a way to control AF carcinogenecity. Moreover, the increasing in glutathione S-transferase activity induced by Z. spina- christi extract may be another way in the prevention of AF carcinogenecity through the increasing of AF-glutathione conjugates and consequently decreased the ability of hepatic microsomes to metabolise AF and preventing the formation of epoxides and DNA adduct

Lee et al. (2001) reported that flavonoids were shown to be potent inhibitors of aflatoxin B₁-8,9-epoxide formation. They found that addition of the flavonoids and flavone strongly inhibited mouse liver microsomal conversion of aflatoxin B₁ to aflatoxin B₁-8,9-epoxide, a metabolically activated mu tagenic product.