Therapeutic ability of some plant extracts on aflatoxin B1 induced renal and cardiac damage

1 Introduction

Aflatoxins are secondary toxic fungal metabolites produced by Aspergillus flavus and A. parasiticus. There are four naturally occurring aflatoxins, the most toxic being aflatoxin B1 (AFB1), and three structurally similar compounds namely aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). Aflatoxins not only contaminate our food stuffs but are also found in edible tissues, milk and eggs after consumption of contaminated feed by farm animals (Bennett and Klich, 2003; Fink-Gremmels, 1999).

Aflatoxins are well known to be potent mutagens, carcinogenic, teratogenic, immunosuppressive and also inhibit several metabolic systems, causing liver, kidney and heart damage. These toxins have been incriminated as the cause of high mortality in livestock and some cases of death in human being (Minto and Townsend, 1997; Wogan, 1999; Bintvihok, 2002; Wangikar et al., 2005; Salunkhe et al., 1987).

The mechanism of AFB1 toxic effect has been extensively studied. It has been shown that AFB1 is activated by hepatic cytochrome P450 enzyme system to produce a highly reactive intermediate, AFB1-8,9-epoxide, which subsequently binds to nucleophilic sites in DNA, and the major adduct 8,9-dihydro-8-(N7guanyl)-9-hydroxy-AFB1 (AFB1 N7-Gua) is formed. The formation of AFB1-DNA adducts is regarded as a critical step in the initiation of AFB1-induced carcinogenesis (Sharma and Farmer, 2004; Klein, 2002; Preston and Williams, 2005).

Although the mechanism underlying the toxicity of aflatoxins is not fully understood, several reports suggest that toxicity may ensue through the generation of intracellular reactive oxygen species (ROS) like superoxide anion, hydroxyl radical and hydrogen peroxide (H₂O₂) during the metabolic processing of AFB1 by cytochrome P450 in the liver. These species may attack soluble cell compounds as well as membranes, eventually leading to the impairment of cell functioning and cytolysis (Towner et al., 2003; Sohn et al., 2003; Berg et al., 2004).

Natural substances that can prevent AFB1 toxicity would be helpful to human and animal health with minimal cost in foods and feed. Traditional medicinal plants were used by some authors for their antifungal, anti-aflatoxigenic and antioxidant activity (Joseph et al., 2005; Kumar et al., 2007).

Green tea (Camellia sinensis, family Theaceae) has been found to possess many health benefits including protection of oxidative DNA damage, lowering atherosclerotic index, and improving blood flow, liver function and oral health. It also has antimicrobial and antioxidant activities (Dufresne and Farnworth, 2001; McKay and Blumberg, 2002; Almajano et al., 2008; Panza et al., 2008).

The functional properties of tea are due to its polyphenols. Tea catechins are the principal tea polyphenols, which are mainly present in green tea and exhibit the most effective antioxidant activity compared to other tea polyphenols. Among tea catechins, epigallocatechin gallate (EGCG) is the most abundant and efficient catechin (Almajano et al., 2008; Panza et al., 2008).

Caraway (Carum carvi L., family, Umbelliferae) is another traditional medicinal plant. The aqueous extract of caraway is used as an aperitif, tranquilizer, carminative, diuretic. The seeds are used to treat ailments such as flatulence, colic pain and bronchitis (Bellakhdar et al., 1997; Lahlou et al., 2007; de Carvalho and da Fonseca, 2006).

Galangal (Alpinia galangal, family, Zingiberaceae) has traditionally been used as spice foods. This spice is, like other spices, rich in phenolic compounds such as flavonoids and phenolic acids which contribute to the overall its antioxidant activities (Juntachote and Berghofer, 2005). Galangal is used for medical purposes such as carminative, stomachic (Yang and Eilerman, 1999) antispasmodic, antiphlogistic and antimicrobial (Viljoen et al., 2003; Oonmetta-aree et al., 2006).

Frankincense (Boswellia serrata, family, Frankincense) is a gum resin secreted by trees of the genus Boswellia of Burseraceae. It has been used for therapeutic purposes as a remedy, especially in the traditional Ayurvedic medicine of India. It was reported that frankincense exerted anti-inflammatory, antidiabetic, antiarthritic, anticancer and immunomodulatory activities in animals (Al-Wadi and Gumaa, 1987; Reddy et al., 1986; Hostanska et al., 2002; Miklaeil et al., 2003).

The active principles, boswellic acids, prevent endotoxin-/galactosamine-induced hepatitis in mice (Safayhi et al., 1991). It also exhibits marked sedative and analgesic effects (Knaus and Wagner, 1996). Boswellia has also been observed to inhibit human leukocyte elastase (HLE), which may be involved in the pathogenesis of emphysema. It also stimulates mucus secretion and thus may play a role in cystic fibrosis, chronic bronchitis and acute respiratory distress syndrome (Safayhi et al., 1991, 1997; Knaus and Wagner, 1996; Rall et al., 1996).

Cinchona (Cenchona officinalis, family, Rubiaceae) is another medicinal plant used for treatment of some diseases. It exhibits antimicrobial activity against bacteria and fungi. The bark of cinchona contains the alkaloid quinine along with several other alkaloids effective against malaria and other parasitic infection, quinovic acid is effective as antitumor and quinidine is used in the treatment of heart diseases (Rojas et al., 2006; Mturi et al., 2002; Trepardoux, 2002; Raffauf et al., 1978; Prinz, 1990).

Methotrexate (MTX) is widely used as a cytotoxic chemotherapeutic agent to treat various neoplastic diseases such as acute lymphoblastic leukemia, lymphoma, solid cancers and autoimmune diseases such as psoriasis and rheumatoid arthritis (Antunes et al., 2002; Brock and Jenning, 2004; Doan and Massarotti, 2005). However, the efficacy of this agent often is limited by severe side effects and toxic sequelae. Since the cytotoxic effect of MTX is not selective for the cancer cells, it also affects the normal tissues.

MTX administration has been reported to cause acute renal failure, hepatotoxicity characterised by necrosis and fibrosis, neurotoxicity and intestinal mucosa oxidative stress (Walker et al., 2000; Sener et al., 2006; Jahovic et al., 2003; Vezmar et al., 2003; Uzar et al., 2006; Miyazono et al., 2004; Cetiner et al., 2005).

Relatively little is known about metabolic alterations occurring in kidney and heart during aflatoxicosis. So the objective of this study is to evaluate the metabolic disorders induced pathology in kidneys and hearts of rats by the exposure to AFB1. Also, the therapeutic benefits of some medicinal plant aqueous extracts in comparison to the currently available anticancer drug, MTX, against these disorders were also investigated.

This can be achieved through measuring some markers of oxidative tissue damage in kidney such as thiobarbituric acid reactive substances (TBARS, index of lipid peroxidation), the antioxidant markers, glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PDH), vitamin C and some indices of energy metabolism in heart [as glycogen, glucose and the glycolytic enzymes, phosphogluco-isomerase (PGI), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH)].

2 Materials and methods

3 Results

Table 1 reveals the abnormal levels of kidney oxidative stress biomarkers in rats that indicate the cellular damage caused by AFB1 treatment. The level of MDA in kidney tissue was markedly increased with concomitant significant decrease in the activities of tissue GR and G-6-PDH as well as in vitamin C level in AFBI-treated animals when compared with normal healthy ones. Administration of different plant extracts after AFB1 injection improve to large extent these parameters levels indicating the therapeutic role of these drugs.

This effect was pronounced in animals treated with green tea (G3), galangal (G5) and frankincense (G7) where these plants modulated most of these altered parameters nearly to their normal levels. However, supplementation of AFB1 treated rats with MTX dramatically amplified the toxic effect of AFB1 where the alterations in the studied parameters were more pronounced in AFB1-MTX treated animals than AFB1-treated ones.

The levels of some markers of energy metabolism in hearts of rats of different experimental groups are shown in Table 2. The result revealed marked decrease in glycogen and glucose contents in the hearts of AFB1 administered animals accompanied with a reduction in the glycolytic enzymes PGI, G3PDH and LDH. Administration of the extracts of plants under investigation, effectively up-regulate the decrease in these energetic biomarkers. But MTX treatment severely aggravated aflatoxicosis induced alteration in cardiac energy supply where the deviation in the studied energetic indices were more evidence in AFB1-MTX treated animals than AFB1-treated ones.

4 Discussion

This study was performed to evaluate the beneficial therapeutic ability of some traditional medicinal plant extracts against mycotoxicosis induced in rat kidneys and hearts by aflatoxin B1. Symptoms of mycotoxicosis are a result of interactions of mycotoxins with functional molecules and subcellular organelles in the animal cell.

Results clearly indicate increase in MDA (index of lipid peroxidation, LPO) in kidneys of aflatoxin-treated rats as compared to control, suggesting kidney oxidative damage. This is in agreement with findings reported previously for rat liver (Abdel-Wahhab and Aly, 2003; El-Gibaly et al., 2003; Liebert et al., 2006; Naaz et al., 2007). As well as liver and kidney in mice (Verma and Nair, 1999; Choudhary and Verma, 2005).

Induction of LPO by AFB1 is considered one of the main manifestations of oxidative damage initiated by reactive oxygen species (ROS) and it has been linked with altered membrane structure and enzyme inactivation. It is exhibited that aflatoxin B1, with the help of microsomal cytochrome p-450 mediated oxidation, is biotransformed into aflatoxin 8-9-epoxide, which is a reactive intermediate and highly toxic (Niki et al., 2005; Sharma and Farmer, 2004; Klein et al., 2002).

Cytochrome p-450 exhibits a key function in the biotransformation of xenobiotics, catalyze the reductive transformation of foreign compounds and displays an oxidase activity resulting in ROS formation (Towner et al., 2003; Sohn et al., 2003). These species trigger cell damage through binding to cell macromolecules as well as membrane, leading to membrane peroxidation which affect the ionic permeability of the membrane and eventually leading to the impairment of cell functioning and cytolysis (Berg et al., 2004).

This is supported by previous investigation stated that aflatoxin is a potent nephrotoxic compound leading to kidney damage and changes in renal function. Also, previous histopathological studies indicated that the exposure to aflatoxin B1 led to sever degenerative renal damage (Wangikar et al., 2005; Dafalla et al., 1986; Gabal and Azzam, 1998; Tassari et al., 2006).

Oxidative damage in the cell or tissue occurs when the concentration of ROS (superoxide radical, hydroxyl radical and hydrogen peroxide) generated exceeds the antioxidant capability of the cell (Sies and Stahl, 1995) or when the antioxidant capacity of the cell decreases. Levels of non-enzymatic antioxidants (vitamin C) and enzymatic antioxidants (GR and G-6-PDH) are the main determinants of the antioxidant defense mechanism of the cell.

The significant reduction in vitamin C level as well as in activities of glutathione reductase (GR) and G-6-PDH (glutathione metabolizing enzymes) in kidneys of AFB1 administered rat could be responsible for increased lipid peroxidation observed during aflatoxicosis. This result is agreed with previous studies which revealed significant reductions in enzymatic and non-enzymatic antioxidants in AFB1-fed rat (El-Gibaly et al., 2003; Choudhary and Verma, 2005).

Vitamin C, which includes ascorbic acid and its oxidation product – dehydroascorbic acid, has many biological activities in human body. It can be defined as an enzyme cofactor, a radical scavenger, and as a donor/acceptor in electron transport at the plasma membrane (Davey et al., 2000).

GR is the key enzyme in the conversion of oxidized glutathione (GSSG) back to the reduced form (GSH). GSH scavenge the electrophilic moieties produced by toxic chemicals and conjugate them to less toxic products (Anilakumar et al., 2004). It also has an important role in the maintenance of vitamin C normal level. During free radical scavenging action, ascorbic acid is transformed into l-dehydroascorbate (Breimer, 1990). GSH is required for the conversion of l-dehydroascorbate back to ascorbate (Preetha et al., 2006). The normal vitamin C level is a therapeutic benefit as it able to reduce oxidative stress through reacting with superoxide radical and hydroxyl radicals (Sies and Stahl, 1995).

So reduction in GR activity may lead to the fall in the level of reduced glutathione and hence decreases the conversion of l-dehydroascorbate to ascorbate and this probably explain the lowered level of ascorbic acid in the aflatoxin-treated animals. Glucose-6-phosphate dehydrogenase (G-6-PDH), a rate-limiting enzyme of the pentose phosphate pathway, is required for NADPH generation which is needed for the maintenance of GSH in its reduced form. Inhibition of G-6-PDH activity may reduce the capacity of the tissue to protect itself from the oxidative stress because less amount of NADPH is produced (Zhang et al., 2000).

Supplementation of different plant extracts, potentially was effective in reducing lipid peroxidation, and enhancing the antioxidant status of kidney in AFB1-treated animals. It is indicated by amelioration of MDA to near normal level and significant improvement the antioxidant defense system (GR, G-6-PDH and vitamin C), suggesting their antioxidant potentials. Two mechanisms can be postulated to the therapeutic effect of the used plant extracts against AFB1 renotoxicity. The first one is that these extracts may exert their beneficial actions through their direct antimicrobial activities. The second mechanism is based on their antioxidant abilities (proved from our data), which may be responsible for protecting the cells against the oxidative stress, possibly by increasing the endogenous defensive capacity of the kidney to combat oxidative stress induced by AFB1.

This emphasizes the fact that herbal medicines often contain multiple active substances which have antimicrobial and antioxidant activities. Some authors reported that several phenolic compounds contribute to the overall antioxidant and antimicrobial activities of herbs against human pathogens The mechanisms of phenolic compounds for antioxidant activity are mainly due to their redox properties and chemical structures, which can act as reducing agents, free radical scavengers or quenchers of the formation of singlet oxygen (Zheng and Wang, 2001; Pizzale et al., 2002; Puupponen-Pimia et al., 2005).

Green tea (G3) was found to posses antimicrobial activity. It also contains polyphenols which have recently been reported to be a potent antioxidant and beneficial in oxidative stress, and to inhibit the initiation of AFB1-induced carcinogenesis in rats and mice. Epigallocatechin gallate, the main ingredient of green tea extract, is a strong chemopreventing of toxic effects of AFB1. It has an important role in converting AFB1 to aflatoxicol which is less toxic, enhancing of glutathione-S-transferase (GST) activity and reducing the metabolic conversion AFB1 to the AFB1-DNA adduct (Almajano et al., 2008; Panza et al., 2008; Chen et al., 2004; Raza and John, 2005; Chou et al., 2000; Ahmed et al., 2002; Tulayakul et al., 2007).

Caraway (G4) is rich in monoterpenes and is known for its antimicrobial activity. In addition, previous studies show that caraway contains potential antioxidant substances such as the flavonoids. Apart from antioxidant properties, caraway also has an important role in reducing cytochrome P450 metabolic activity. Thus this may reduce the metabolic conversion of AFB1 to the highly reactive intermediate, AFB1-8,9-epoxide which has the major role in AFB1 toxicity and carcinogenicity (Preston and Williams, 2005; Iacobellis et al., 2005; Satyanarayana et al., 2004; Naderi-Kalali et al., 2005).

Galangal (G5) was found to have antimicrobial effect which is related to the presence of several components acted as antimicrobial agents. 1,8-Cineole, the main component of galangal in the essential oil, has been previously reported to have an antibacterial activity. It has a mechanism of action against bacterial cells involving disruption of cytoplasmic membrane and coagulation of cell contents. Also, several researchers have reported that galangal showed antioxidant activity in a model system (Viljoen et al., 2003; Oonmetta-aree et al., 2006; Gachkar et al., 2007; Cheah and Abu-Hasim, 2000; Javanmardi et al., 2003).

Frankincense (G6) oils was reported to exhibited antibacterial and antifungal activities. The water-soluble fraction of the frankincense show hepato-protective and reno-protective property. It possess antioxidant activity as it has the ability to inhibit induced nitric oxide (NO) and inflammatory mediators production under in vivo and in vitro condition (Umez, 2000; Pandey et al., 2005; Gayathri et al., 2007).

Cinchona (G7) (Cinchona officinalis) was also exhibited antimicrobial activity against bacteria and fungi which may related to the alkaloids (namely, quinine, quinidine, cinchonine and cinchonidine) and steroids found in this plant (Rojas et al., 2006).

Administration of the anticancer drug, MTX, to AFB1-treated rats (G8) dramatically amplified the toxic effect of AFB1 induced in kidney which was documented by elevated MDA level with concomitant decrease in antioxidant defense systems, vitamin C, GR and G-6-PDH compared with normal animals. The behavior implicates the presence of oxidative tissue damage. These deviations were more evidence in AFB1-MTX treated animals than in AFB1-treated group.

This result is agreed with previous authors who reported that administration of MTX induces lipid peroxidation accompanied by significant reductions in antioxidant defense mechanism in different tissues including kidney, which is considered an important cause of destruction and damage to cell membranes and has been suggested to be a contributing factor to the development of MTX-mediated tissue damage (Cetiner et al., 2005; Rajamani et al., 2006). The significant reduction in the glutathione metabolizing enzymes (GR and G-6-PDH) as well as vitamin C induced by MTX may be explained by previous studies demonstrated that the cytosolic NAD(P)-dependent dehydrogenases are inhibited by MTX, suggesting that the drug could decrease the availability of NADPH in cells which is used by glutathione reductase to maintain the cytosolic antioxidant glutathione in reduced state (Cetiner et al., 2005; Caetano et al., 1997). Consequently, this may lead to a reduction in the effectiveness of the antioxidant vitamin C (Babiak et al., 1998).

Concerning with the effect of AFB1 administration on some energy metabolism biomarkers in rat hearts, the results revealed that marked decrease in glycogen and glucose contents of animal hearts in response to aflatoxicosis accompanied by a decrease in the glycolytic enzymes, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glucose isomerase (GI) and lactate dehydrogenase (LDH), suggesting that AFB1 may interfere with the cellular energy supply through its inhibitory action on glycogen synthesis, glucose uptake (a decrease in glucose transport from blood to myocardial cells) and glycolysis in heart. Similar result was obtained by Tessier et al. (2003) who reported that cardiac glycolysis and glycogen synthesis are impaired by a bacterial endotoxin in rats. Our results are consisted with some authors who reported that AFB1 decreases tissue glycogen by inhibiting the activities of biosynthetic enzymes and stimulating glycogenolysis through inhibiting intracellular cyclic adenosine monophosphate (cAMP) hydrolysis, which responsible for phosphorylase activation, the rate-limiting enzyme for glycogen (Kiessling, 1986; Bonsi et al., 1999).

Also it was found that AFB1 reduces glucose transport and affects some enzymes engaged in glucose metabolism in the liver. The reduction in glucose uptake may be attributed to the decrease in the number of GLUT 1 and GLUT 4 transporters in the sarcolemma in response to aflatoxicosis. These proteins are normally stored in cytoplasmic membranes and can be recruited to the plasma membrane as needed (Kiessling, 1986).

The loss of cardiac anaerobic glycolysis under the effect of AFB1 toxicity may be ascribed to the low level of intracellular glucose, in addition to the decrease in LDH activity may affect the re-generation of NAD from NADH, the oxidizing equivalents necessary to GAPDH, which sustains the continuous operation of glycolysis and hence the rate of glycolysis will be reduced (Brooks et al., 2003).

On the other hand, these enzymes may be released from cardiac muscle fibers due to aflatoxicosis causing tissue damage. The later suggestion is confirmed by histopathological investigations proved that gross cardiac anomalies due to AFB1 indicated by dilation of the heart and fusion of auriculo-ventricular valve leading to obstruction of passage of the blood from auricles to ventricles, vaculations and degeneration of cardiac muscle fibers (Liebert et al., 2006).

Glycogen utilization through glycolysis is one of the metabolic pathways which directly participate in generation of sufficient adenosine triphosphate (ATP) to meet energy demands of the heart. The myocyte requires continuos production of ATP to survive because it cannot be stored. Alterations of such bioenergetic sources which have a crucial role in cellular protection may render the heart susceptible to metabolic stress and affect the myocardial contractile performance, leading heart failure. This is supported by some authors who reported that exposure to aflatoxin B1 toxicity caused heart defects (Carvajal and Sánchez, 2003; Mancini et al., 1999; Pasha et al., 2007).

Administration of different plant extracts, effectively modulate the lowered levels of glycogen and glucose as well as the activities of the key enzymes involved in glucose metabolism in hearts of rats in response to aflatoxicosis. Modulation of the heart energy metabolism by the used drugs could be beneficial to the amelioration of events leading to cardiopathy. No comparable data are so far available for the effect of these plants on the heart energy metabolism, however, the possible mechanisms which may explain this good effect of the tested different plant extracts are that they may have a crucial role in inducing the enzymes implicated in glycogen synthesis and have the ability to increase glucose uptake by modulating the alterations in the glucose transporter GLUT in the cardiac muscle affected by AFB1. Also, the amelioration of glycolytic enzyme activities may attributed to the availability of glucose, in addition these extracts may have a direct beneficial action in cardiac tissue repair due to their antioxidant abilities as previously mentioned, thus preventing enzymes release.

Administration of MTX to AFB1-treated animals aggravates the cardiotoxic effect of AFB1 via decreasing the energy metabolic markers in the animal hearts which was ensured by the reduction in the glucose and glycogen contents of the heart with concomitant decrease in the tested glycolytic enzymes. These alterations were more severe in AFB1-MTX treated rats than in AFB1-treated group. The decrease in the glucose by MTX may ascribed to its ability to decrease glucose transport into the cells as one of its mechanism of actions against tumor cells. This finding is supported by previous authors who declared that MTX inhibited glucose uptake by Ehrlich ascites cells (Fung et al., 1986). The decrease in glycogen may attributed to either the inhibitory action of MTX on glycogen synthesis or the reduction of intracellular cardiac glucose, the substrate for glycogen synthesis. The decrease in glycolytic enzymes may be resulted from either the decrease in cardiocyte glucose content or the release of these enzymes due to tissue damage. This is ensured by some authors reported that treatment of rat hepatocyte with MTX for 24 h resulted in increase in enzyme leakage supporting the conclusion that MTX was cytotoxic agent in rat hepatocyte (Walker et al., 2000). Our data collectively support the hypothesis that cellular oxidative stress is unpredictable side effects of MTX and may consider a critical step in MTX-mediated injury (Sener et al., 2006).

In conclusion, our results have shown that all aqueous extracts of the examined plants alleviate AFB1 induced kidney oxidative stress and alteration of cardiac energy metabolism and can be regarded as good therapeutic agents against aflatoxicosis.