Ameliorating Effect of Phytoestrogens on CCl₄-Induced Oxidative Stress in the Livers of Male Wistar Rats

Abstract

Glutathione-S-transferases and glutathione play a key role in the detoxification of most toxic agents. In the present study, the protective effects, if any, of isoflavone phytoestrogens—genistein and daidzein on the carbon tetrachloride (CCl₄) induced changes in the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione S transferase (GSH) and levels of glutathione (GSH) and thiobarbituric acid reactive substances (TBARS)—were studied. The activities of ALT and AST were assayed in the serum, whereas the activity of GST and levels of GSH and TBARS were determined in the livers of rats. The current study involved the division of animals into two main groups: (i) rats pretreated with genistein and daidzein for three days; and (ii) non-pretreated rats. In the pretreated group, rats received oral doses of genistein (7.9 µmol/kg body weight) and daidzein (7.9 µmol/kg body weight) for three consecutive days (once daily) followed by oral dose of CCl₄ on the 4th and the 5th day concurrently with the phytoestrogens-genistein or daidzein. In the non-pretreated group animals received oral dose of CCl₄ (1 ml/kg body weight) for two consecutive days along with the phytoestrogens-genistein or daidzein. Treatment of male rats with CCl₄ significantly elevated the activity of ALT and AST in serum and levels of TBARS in the liver. On the other hand, CCl₄ resulted in decreased activity of GST and lowered the GSH levels. Coadministration of genistein and daidzein with CCl₄ could not restore the alterations in the activity of ALT and AST caused by CCl₄ to normal control levels. However, repeated dose treatments with genistein and daidzein for three days prior to the administration of CCl₄ restored such alterations to normal levels. Our results indicate that genistein is more effective than daidzein in counteracting the inhibition of GST activity caused by CCl₄ and restoring it to normal levels. Genistein was also more effective than daidzein restoring the induced TBARS levels caused by CCl₄ to normal control levels when rats were pretreated with the isoflavone orally for three days. It has been observed that the tested isoflavonoids were able to antagonize the toxic effects of CCl₄. Such counteracting effects were more pronounced for genistein and when the phytoestrogens were administered as repeated doses prior CCl₄ administration.

INTRODUCTION

Conjugation of toxic metabolites with glutathione (GSH), catalyzed by glutathione S-transferases (GST), is one of the major pathways for the detoxification of toxic metabolites [[1]]. Previous studies have shown that GSH and GST can reduce the covalent binding of epoxides of well-known carcinogens, e.g. benzo(α)pyrene and aflatoxin B1 to DNA and consequently reduce hepatocarcinogenesis caused by these compounds [[2-4]]. Several dietary compounds have been demonstrated to reduce gastrointestinal cancer rates in both human and animals through induction of GST activity [[5]]. The coffee–specific diterpenes cafestol and kahweol have been reported to be anticarcinogenic in several animal models due to induction of glutathione S- transferase π class [[6]].

Carbon tetrachloride has been extensively used to elicit experimental liver damage in rats. It is a widely accepted animal model remarkably similar to human alcoholic cirrhosis both histologically and systemically. CCl₄ induced liver damage has been thought to depend on the formation of reactive intermediates such as trichloromethyl radical produced by cytochrome P450 mixed function oxidase system and further converted to a peroxy radical [[7], [8]]. These free radicals react with polyunsaturated fatty acids to propagate a chain reaction leading to lipid peroxidation or bind covalently to lipids and proteins, resulting in the destruction of membranes. Peroxidation of the polyunsaturated fatty acids in biological membranes is believed to be the mechanism of toxicity for some chemicals such as carbon tetrachloride, ethanol, etc. Furthermore, CCl₄ induced liver damage is lowered by substances that scavenge free radicals [[9-11]]. It is thought that antioxidants play a significant role in protecting the living organisms from the toxic effects of chemical substances such as CCl₄ and carcinogens. It has been reported that compounds possessing reactive phenolic groups are endowed with antioxidant properties in vitro [[12-14]]. This is the case for the natural flavonoids [[15]], inhibiting different lipid peroxidation systems [[16-20]]. Many different natural compounds could alleviate the toxicity of CCl₄ and other toxic agents through inhibition of lipid peroxidation, which is generated from CCl₄ metabolism [[21]]. For example, Vitamin E, d-α-tocopherol, is the most important endogenous lipid soluble antioxidant and is thought to play a major role in protecting cellular membranes against lipid peroxidation and associated loss of protein sulfhydrl groups [[22], [23]]. Also, pretreatment of rats with Vitamin E has been found to protect brains against the toxicity of CCl₄ [[24]].

Soybeans, which have long been part of the diet in Asian countries, are enriched with phytoestrogens [[25]]. Epidemiological studies have shown that consumption of soybeans decreases the risk of various diseases and conditions including breast cancer [[26]], prostrate cancer [[27]], colon cancer [[28]], osteoporosis [[29]], menopausal symptoms [[30]], and coronary heart disease [[31]]. The representative isoflavones in soybeans are genistein and daidzein (Fig. 1). Soy isofavones have been reported to have a variety of biological activities including estrogenic [[32]], antioxidative [[33]], antiosteoportic [[34]] and anticarcinogenic [[35]]. Genistein, in particular, inhibits the proliferation of a variety of tumor cell lines in culture [[36]] and is thought to be the most effective isoflavone in cancer treatment and prevention.

short-legendFigure 1

Considering the multifaceted role played by these phytoestrogens (genistein and daidzein) the present study was, therefore, conducted to investigate the changes in the activities of ALT, AST, GST and the levels of GSH and TBARS and to evaluate the protective effect of these antioxidants against the toxicity of CCl₄ in various treatment strategies.

MATERIALS AND METHODS

Chemicals

Carbon tetrachloride, Bovine Serum Albumin (BSA), 1-chloro-2, 4-dinitrobenzene CDNB), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), sodium dithionate, nicotinamide adenine dinucleotide phosphate reduced (NADPH), 2-thiobarbituric acid (TBA), glutathione reduced (GSH), etc. were obtained from Sigma Chemical Co., St. Louis, USA. All other chemicals used were of the highest available purity grade.

Animals and Administration Schedule of CCl₄ and Phytoestrogens (Genistein and Daidzein)

Male wistar rats weighing 150–180 g obtained from our laboratory-maintained colony were used as experimental models throughout the study. The local ethics committee approved the design of the experiment and the protocol conformed to the laid guidelines. Animals were housed in cages where food and water were provided ad libitum. Table 1 summarizes the protocol of administration of carbon tetrachloride and phytoestrogens (Genistein and Daidzein). Briefly, male wistar rats were orally fed carbon tetrachloride (1 ml/kg bwt) and phytoestrogens (genistein and daidzein) (7.9 µmol/kg bwt) independently as well as in combination. The control animals received equivalent amount of saline.

Table 1. Animal treatment protocol

Experimental group	Day 1	Day 2	Day 3	Day 4	Day 5
Group I	Saline	Saline	Saline	Saline	Saline
Group II	Saline	Saline	Saline	CCl4	CCl4
Group III	Saline	Saline	Saline	Genistein	Genistein
Group IV	Saline	Saline	Saline	Daidzein	Daidzein
Group V	Saline	Saline	Saline	CCl4 + Genistein	CCl4 + Genistein
Group VI	Saline	Saline	Saline	CCl4 + Daidzein	CCl4 + Daidzein
Group VII	Genistein	Genistein	Genistein	CCl4 + Genistein	CCl4 + Genistein
Group VIII	Daidzein	Daidzein	Daidzein	CCl4 + Daidzein	CCl4 + Daidzein

All animals were sacrificed on day 6 after 24 hrs of last treatment. The number of rats in each group was 6.

ENZYMES ASSAY

Animals were sacrificed by cervical dislocation at the designated time point. Freshly removed livers were washed and homogenized in three volumes of 0.1 M potassium phosphate buffer, pH 7.4. The homogenates were centrifuged at 11,000 × g for 20 min at 4°C. The supernatants were used for the estimation of glutathione S-transferase, glutathione levels and thiobarbituric acid reactive susbstances. The supernatant fractions were centrifuged at 105,000 × g for 1 hour at 4°C to yield a microsomal pellet, which was then resuspended in 0.1 M phosphate buffer at H 7.4. Blood was taken out from the heart for serum study. Lipid peroxidation was estimated in the liver by quantitation of thiobarbituric acid reactive substances (TBARS) and was assayed by the method of Wright et al. (6). The absorbance was read at 535 nm for estimation of malonaldehyde (MDA) concentration. The results were expressed as the nmol MDA formed/hr/g tissue at 37°C using a molar extinction coefficient of 1.56 × 10⁵ M⁻¹cm⁻¹. Reduced glutahione was assayed by the method of Jollow et al. [[7]]. Glutathione S-transferase activity was measured by the method of Habig et al. [[8]]. The changes in absorbance were recorded at 340 nm and enzyme activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6 × 10³ M⁻¹cm⁻¹. Protein concentration was estimated by the method of Lowry et al. [[14]]. ALT and AST were evaluated using commercially available kits following the manufacturer's instructions.

STATISTICS

Figures in Table 2 indicate mean value±SEM. Differences between control and treated animals were analysed using student's t-test taking P < 0.05 as significant.

Table

Groups	Treatment	GST (nM/min/mg)	GSH (µM/g liver)	LPO (nM/g liver)	ALT (K.U)	AST (K.U)
I	Control	0.9 ± 0.04	8 ± 0.9	230 ± 15.3	13.3 ± 0.7	14.3 ± 0.5
II	CCl4	0.36 ± 0.02	1.6 ± 0.18	506 ± 33.66	42 ± 1.2	39 ± 1.0
III	Genistein	1.03 ± 0.05	9.60 ± 1.08	221.5 ± 14.7	15.2 ± 0.8	14.9 ± 0.7
IV	Daidzein	0.77 ± 0.03	7.28 ± 0.82	251.16 ± 16.71	18.5 ± 0.8	20.9 ± 0.7
V	CCl4 + Genistein	0.71 ± 0.03	5.20 ± 0.59	333.50 ± 22.19	30.2 ± 1.0	32.6 ± 0.9
VI	CCl4 + Daidzein	0.59 ± 0.03	4.20 ± 0.31	391.00 ± 26.01	38.7 ± 1.0	35.6 ± 1.0
VII	CCl4 + Genistein	0.79 ± 0.03	6.55 ± 0.72	249.08 ± 19.89	18.1 ± 0.9	17.8 ± 0.7
VIII	CCl4 + Daidzein	0.63 ± 0.03	4.40 ± 0.50	329. ± 23.2	36.7 ± 0.9	33.3 ± 0.7

*Animals in these groups were pretreated with genistein and daidzein (Group VII and VIII respectively) for 3 days prior to CCl₄ administration.

Values represent means±SEM for 6 animals.

RESULTS

The Importance of nutrition in protection of living organisms from the toxic effects of chemical and environmental carcinogens has recently been realized. Components of diet like carbohydrates, proteins, vitamins and lipids are important in maintaining levels of various enzymes required in the body's defense system for providing protection against carcinogens. The beneficial effect of antioxidants against chemicals and carcinogens has also been explained on the basis of the fact that they activate the major stalwarts of the detoxification system- the glutathion-S-transferases. In consonance with these observations, our results indicate that treatment of rats with phytoestrogens enhanced the activity of GST relative to the CCl₄ treated group. This increase of GST activity can be speculated as one of the protective mechanisms of phytoestrogens, in particular genistein, against the toxic metabolites of CCl₄. GST activity was markedly inhibited by CCl₄ administration. The hepatic GST activity decreased by ∼60% as compared to control on CCl₄ administration. Treatment with genistein alone increased the hepatic GST activity by ∼25% but daidzein administration showed a decrease in hepatic GST activity by ∼32%. However, when CCl₄ and genistein were given simultaneously a decrease of ∼32% in hepatic GST activity was observed. Coadministration of CCl₄ with daidzein caused a decrease in hepatic GST activity of the order of ∼35%.

Biochemical analysis of CCl₄ intoxication (Group II) showed a significant increase (p < 0.05) in the serum markers of liver damage (ALT and AST). It is evident from the serum ALT and AST data that rats receiving genistein along (Group III) did not exhibit any liver injury whereas daidzein treated rats (Group IV) did show slight perturbations of ALT and AST activity from control levels. The animals receiving genistein and CCl₄ (Group V) showed an alleviation of the damage elicited by CCl₄ though the serum enzymes activities were still high as compared to control levels. The protective effect was more pronounced in case of Group VII, wherein rats were pretreated with genistein for three days prior to CCl₄ administration. In contrast to the almost complete recovery from serum enzyme elevations in Group VII where rats received pretreatment with genistein, recovery was not notable in the daidzein-pretreated group. This establishes that pretreatment of rats with genistein was able to counteract the CCl₄ induced liver damage more effectively in comparison to daidzein. It may be speculated that the dose of 7.9 umol/kg bwt of daidzein was not enough perhaps to prevent liver damage induced by CCl₄ in cotreatment and pretreatment strategies.

Glutathione is an important cellular factor influencing the effectiveness of a variety of chemotherapeutic and alkylating agents: organs with low GSH levels are more susceptible to the action of these agents, whereas those with high GSH levels are more resistant. In accordance with these observations, the mechanism of genistein protection against the toxicity of CCl₄ might be partly due to the enhancing of GSH levels, since we have observed that Group III rats that were only genistein fed had GSH levels higher than Group I control rats. Moreover, the protective effect was significant in Group VII animals where genistein administration as repeated oral doses for three days prior to CCl₄ subjection counteracted the perturbations of GSH levels caused by CCl₄. This was not the case in daidzein treated rats (Group VIII), where daidzein pretreatment could restore the perturbed GSH levels by 50% only. Comparison of pretreated (Group VII and VIII) and the cotreated groups (Group V and VI) highlight that pretreatment strategy is more effective in offering cellular protection.

Group II animals that were CCl₄ fed showed markedly increased TBARS levels. In Groups II and IV, wherein animals were fed genistein and daidzein alone at a dose level of 7.9 µmol/kg body weight, TBARS levels were not perturbed significantly. Simultaneous treatment of CCl₄ and genistein (Group V) showed increased TBARS levels as compared to controls but they were ameliorated as compared to the CCl₄ trated group. The extent of alleviation of the induced TBARS levels was less in Group VI (daidzein cotreated) as compared to Group V (genistein cotreated). Interestingly, pretreatment of rats with genistein as repeated doses for 3 days was found to significantly counteract the induced level of TBARS caused by CCl₄ to almost control levels. In our model of CCl₄ induced liver injury, we observed that genistein pretreatment could effectively alleviated the CCl₄ induced TBARS levels. A similar counteraction was observed in Group VIII animals (daidzein pretreated), which was to a lesser extent than the genistein pretreated Group VII animals. A comparison of Group VI and VIII again potentiates, the fact that pretreatment strategy was more effective in counteracting the CCl₄ induced liver injury.

DISCUSSION

Natural flavonoids possess reactive phenolic groups and show antioxidative properties in vitro. In agreement with this, we have chosen isoflavonoids, genistein and daidzein, which bear structural similarlity to flavonoids, and there is a likelihood that the protective influence of genistein can be explained by the chain breaking activity of polyphenols, which act as hydrogen-atom donors to the peroxy radical involving termination of radical chain reactions in addition to the possible scavenging of activated oxygen species at the stage of initiation. Since phenolic compounds when administered to animals can undergo extensive metabolism modifying their activity, it was of interest to assess the effect of these isoflavonoids—genistein and daidzein—on animal models associated with lipid peroxidation. This was kept in mind when choosing the carbon tetrachloride system, since the oxidative stress developed in this system is primarily due to extensive lipid peroxidation.

Reduced glutathione is one of the most important scavenger against oxidative damage. It may act not only as a scavenger by itself but also through some GSH dependent enzymes. Reports have indicated that the threshold for the onset of lipid peroxidation to occur appeared to be at a GSH concentration of about 25% of the peak physiological concentration. The reasons for glutathione depletion may be multifactorial. For example, electrophilic products from lipid peroxidation can spontaneously react with GSH to form conjugates [[44]] or, alternatively, the hepatic transulfuration pathway may be impaired in cirrhosis [[45]], which could hinder the conversion of methionine to cysteine, a process that is necessary for glutathione systhesis. Finally, the destruction of hepatic cells themselves could contribute to GSH depletion. Our results indicate the pretreatment of animals with genistein probably strengthens the antioxidant status of animals by markedly increasing the GSH levels. Thus group VII and group VIII animals showed less vulnerability to the toxic action of CCl₄, possibly due to the scavenging of CCl₄ metabolites by enhanced GSH levels. Induction of GSH levels may be due to the enhancing of GSH synthesizing enzymes such as γ-glutamyl cysteine synthetase and GSH synthetase, which are the key enzymes in biosynthesis of GSH [[46], [47]]. We also speculate that genistein may cooperate with the physiological defense molecules such as reduced glutathione in such a way as to protect animals against oxidative stress.

We observed that genistein administration resulted in increased GSH levels. One possible mechanism for the increased GSH level observed in tissues during genistein treatment may be the activation of enzyme(s) involved in de novo biosynthesis of GSH. Under normal conditions a significant portion of GSH is generated through the reduction of oxidized GSH by gluatathione reductase and the remaining GSH is produced by the biosynthetic pathway. However, during CCl₄ mediated stress, when the demand for GSH increases, the pathway of GSH regeneration through de novo biosynthesis becomes more prominent. This is especially true for liver, which can generate cysteine from methionine for GSH biosynthesis and can rapidly export GSH for supplying extrahepatic tissues [[48]]. The exzyme, γ-glutamyl cysteine synthetase, is recognized as the rate-limiting enzyme for GSH synthesis and it is known to be regulated by the concentration of GSH [[49]].

Our results revealed that when CCl₄ stress was imposed on genistein pretreated animals (Group VII), the marked decrease of GSH levels was counteracted to an appreciable extent. It is likely that activity of γ-glutamyl cysteine synthetase may be stimulated by genistein to maintain the level of GSH in the normal range. However, this is not the case with daidzein pretreated animals, where we observed that the GSH levels were not restored significantly. It may be possible that the net effect may overwhelm the compensatory mechanism for meeting the excess demand for GSH.

The hepatotoxic effects of CCl₄ are thought to result from its reductive dehalogenation by cytochrome P450 into its trichloromethyl free radical. This radical quickly adds molecular oxygen to form the trichloromethyl peroxyl radical. Abstraction of hydrogen atoms from unsaturated lipids by such radicals creates carbon centered lipid radicals. These lipid radicals quickly add molecular oxygen to form lipid peroxyl radicals, thereby initiating the process of lipid peroxidation. Unless scavenged by antioxidants or other radical scavengers, these lipid peroxyl radicals in turn abstract hydrogen atoms from other lipid molecules, thereby propagating the process of lipid peroxidation. Membrane deterioration (and leaking of cytosolic and membrane enzymes) due to extensive stimulation of lipid peroxidation could be an important facet of CCl₄ toxicity and the observed inhibition of lipid peroxidation by genistein may account for its beneficial action. Theoretically, genistein may act in this model by upregulating the GST system, which aids detoxification by scavenging the free radicals or possibly by decreasing the metabolic activation of CCl₄. The latter issue still needs to be addressed. This protective action of genistein is probably associated with its antioxidant properties, possibly acting as a free radical scavenger. Genistein pretreatment produced an almost complete inhibition of hepatic lipid peroxidation in CCl₄ treated as well as a general amelioration of the severity of hepatic injury as evidenced by restored ALT and AST activities. However, we were unable to demonstrate an inverse relationship between glutathione depletion and an increase in lipid peroxidation products. This lack of an inverse relationship is confounded by the observation that phytoestrogen treatment did not produce a significant improvement of hepatic GSH content as compared to the normalization of lipid peroxidation. At issue is how genistein pretreatment could minimize lipid peroxidation if the existing glutathione content was not significantly improved upon. Plasma membrane stabilization and inhibition of hepatic cytochrome activity can be postulated as plausible mechanisms contribution to the hepatoprotection against lipid peroxidation.

Glutathione and glutathione S-transferases help in the protection of cells from the deleterious effects of toxic and carcinogenic compounds [[50]]. Inducers of GSTs are generally considered as protective compounds against carcinogens. The chemoprotective activity of antioxidants against carcinogens has been explained on the basis of the fact that they activate the detoxification system such as GSTs. The human diet is composed of many components that arrest various steps of the carcinogenic process [[51]]. IN consonance with these observations, prior treatment of male rats with genistein and daidzein enhanced the activity of GST relative to CCl₄ treate group (Group I). This enhancement of GST activity can be speculated as one of the protective mechanism of soy isoflavones against the toxic metabolites of CCl₄. Supporting this suggestion, it has been reported that genistein and daidzein inhibit low density lipoprotein oxidation, thereby potentiating the efficacy of phytoestrogens as effective antioxidants. On the contrary, CCl₄ treatment to Group II animals for 48 hrs significantly inhibited the activity of GST in the livers of rats. This finding may be speculated as an inhibitory effect of GSH conjugates on GST activity. Another plausible explanation, as has been suggested by a recent in vitro study [[52]], would be that of a direct covalent modification of the enzyme structure by lipid peroxidation products.

In conclusion, the present study shows that CCl₄ induced oxidative stress produces a decrease in the hepatic GST activity and a decline in the hepatic GSH antioxidant system. The significant impairment of this hepatoprotective system was related to a substantial increase in the free radical production. Phytoestrogen treatment resulted in a general amelioration in the degree of hepatic injury, as evidenced by the amelioration of the ALT, AST activities, together with the normalization of lipid peroxidation, but this could not be ascribed solely to a significant improvement of the hepatic GSH antioxidant system. The beneficial effects were more pronounced with genistein, in particular, and when the phytoestrogens were administered as repeated doses three days prior to CCl₄ administration.