Mistletoe alkali inhibits peroxidation in rat liver and kidney

Abstract

AIM: To explore the antioxidant and free radical scavenger properties of mistletoe alkali (MA).

METHODS: The antioxidant effect of mistletoe alkali on the oxidative stress induced by carbon tetrachloride (CCl₄) in rats was investigated. The rats were divided into four groups (n = 8): CCl₄-treated group (1 mL/kg body weight), MA -treated group (90 mg/kg), CCl₄+MA-treated group and normal control group. After 4 wk of treatment, the level of malondialdehyde (MDA), a lipid peroxidation product (LPO) was measured in serum and homogenates of liver and kidney. Also, the level of glutathione (GSH), and activities of glutathione reductase (GR), glutathione peroxidase (GSPx), superoxide dismutase (SOD), and glutathione-S-transferase (GST) in liver and kidney were determined. Scavenging effects on hydroxyl free radicals produced in vitro by Fenton reaction were studied by ESR methods using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap reagent and H₂O₂/UV as the OH· source. Urinary 8-hydroxydeoxyguanosine (8-OHdG) was determined by competitive ELISA.

RESULTS: In CCl₄-treated group, the level of LPO in serum of liver and kidney was significantly increased compared to controls. The levels of GSH and enzyme activities of SOD, GSPx and GR in liver and kidney were significantly decreased in comparison with controls. In CCl₄+MA-treated group, the changes in the levels of LPO in serum of liver and kidney were not statistically significant compared to controls. The levels of SOD, GSPx and GR in liver and kidney were significantly increased in comparison with controls. There was a significant difference in urinary excretion of 8-OHdG between the CCl₄-treated and MA-treated groups.

CONCLUSION: Oxidative stress may be a major mechanism for the toxicity of CCl₄. MA has a protective effect against CCl₄ toxicity by inhibiting the oxidative damage and stimulating GST activities. Thus, clinical application of MA should be considered in cases with carbon tetrachloride-induced injury.

Key Words: Mistletoe alkali; Inhibition of peroxidation; Free radical; Liver and kidney

INTRODUCTION

Recently, great attention has been focused on the role of the antioxidative defense system in oxidative stress. Endogeneous antioxidants in medicinal herbs may play an important role in antioxidative defense against oxidative damage, possibly protecting the biological functions of cells[1]. There is increasing interest in the protective biological function of natural antioxidants contained in Chinese medicinal herbs, which are candidates for the prevention of oxidative damage[2,3].

Viscum coloratum (Komar.) Nakai has long been categorized as a traditional Chinese medicine in China, USA and other countries. A number of lipid-soluble antioxidants have been isolated from Viscum coloratum (Komar.) Nakai, such as mistletoe alkali, thionins, glucoprotein, polysaccharose[4,5]. In the present study, we investigated the antioxidant activities of mistletoe alkali using a rat model of carbon tetrachloride (CCl₄)-induced hepatotoxicity and nephrotoxic nephritis, which could assess in vivo antioxidant activities of Chinese medicinal herbs.

Lipid peroxidation is one of the reactions induced by oxidative stress and is especially active in tissues rich in polyunsaturated fatty acid. There have been few comparative studies on the antioxidant ability of tradi-tional Chinese medicine to modify the susceptibility of organs or tissues to oxidative stress and to alter the cellular antioxidant defense system[6,7]. We have recently found that some compositions of Viscum coloratum (Komar.) Nakai can act effectively in vitro as antioxidants and pe-roxyl radical scavengers. Mistletoe alkali is one of these antioxidant compositions.

As the liver is the main target organ of CCl₄, and the kidney is the main site of CCl₄ accumulation, the present study was to investigate the oxidative status of both organs in rats simultaneously exposed to CCl₄. To determine the ability of mistletoe alkali to act as antioxidants in vivo, we fed rats with mistletoe alkali (90 mg/kg per day) and examined the metabolism of this compound as well as its effects on oxidative stress in vitro.

MATERIALS AND METHODS

Mistletoe alkali preparation

Viscum coloratum (Komar.) Nakai was immersed into the acidity aqueous solution and smashed to particles, and then the alkaloid was taken out by precipitation with alkaline. In brief, Viscum coloratum (Komar.) Nakai was smashed to particles and soaked in the acidity aqua for 48 h, the liquid was added to the alkali to precipitate and remove infusible part and the alkali was added again to precipitate its total alkali. The acidity aqua of Viscum coloratum (Komar.) Nakai was proceeded to precipitate the different alkalinity with the aqua.

CCl₄-induced hepatotoxicity and nephrotoxic nephritis

Female Sprague Dawley rats (200-250 g) were maintained in a 12-h light/dark cycle at 22°C with free access to food and water. Vitamin E was used as positive control. In each experiment, the animals were randomly assigned into groups (n = 8). In the pretreatment groups, the animals were treated intragastrically with the drug preparation at a desired daily dose for 6 d. Mistletoe alkali and vitamin E were dissolved or suspended in olive oil. Twenty-four hours after the last dosing, the animals were administered intragastrically with CCl₄ (11% (v/v) in olive oil at a dose of 1 mL/kg. Control animals were given the appropriate vehicle, and all treatments were conducted between 900 and 1000 h to minimize variations in animal response due to circadian rhythm. The animals were killed 24 h after CCl₄ treatment. After 2 wk of mistletoe alkali treatment, the rats were starved for 12 h and killed by anesthetization with diethyl ether. Blood was taken from the abdominal aorta and plasma was separated. The kidneys, liver, stomach, large and small intestines were removed and washed with cold PBS. Plasma and tissue homogenates were stored at -80°C. Protein concentrations were determined with bicinchonic acid (BCA) protein assay kit (Pierce, Rockford, IL) with bovine serum albumin as the standard.

Chemicals

Vitamin E, 5,5-dimethyl pyrrole-N-oxide (DMPO), thiobarbituric acid ( TBA) were bought from the Sigma Company (USA). “8-OhdG Check” kit was purchased from Fukuroi (Japan) for the control of aging. All other chemicals were of analytical grade.

Hydroxyl radical scavenging assay by electron spin resonance trapping method

Hydroxyl radical (· OH) scavenging activity of mistletoe alkali was determined by spin trapping ESR using DMPO as a spin trap reagent as previously described[8]. Both Fenton reaction and H₂O₂/ UV system were used as the · OH source. Comparative study on the · OH scavenging activity of mistletoe alkali was carried out in the presence of liver and kidney homogenates and serum using the Fenton system as · OH generator. Briefly, rat liver and kidney homogenates (0.923 mg protein) were incubated with an aliquot of mistletoe alkali at 37°C for 30 min. The homogenates (100 mL) were mixed with 160 mL of distilled water, 30 mL of 100 mol/L H₂O₂ and 30 mL of 200 mol/L DMPO. Then, 30 mL of 2 mmol/L FeSO₄ was finally added to the mixture to initiate the Fenton reaction. DMPO-OH adduct formation was measured in a silica flat cell 2 min after the addition of FeSO₄ using a JEOL JESTE 200 electron spin resonance (ESR) spectrometer (X-Band Microwave Unit). The spectrometer settings were as follows: microwave power: 8 mW; microwave frequency: 9.20 GHz; modulation amplitude: 0.1 mT; time constant: 0.03 s; sweep time: 30 s; center fields: 332.6/322.6 mT.

TBARS formation determination

Briefly, an aliquot of liver and kidney homogenates was mixed with 0.2 mL of 8.1% SDS, 1.5 mL of 20% acetic acid and 1.5 mL of 0.8% TBA, then the volume was adjusted to 4.0 mL with distilled water. After boiled at 95°C for 60 min, the reaction solution was extracted with 1.0 mL of distilled water and 5.0 mL of n- butanol and pyridine (15:1 v/v). The absorbance at 532 nm of the organic layer was determined after centrifugation. The whole procedure was performed as previously described[9].

GSH-Px activity determination

Briefly, an aliquot of liver and kidney homogenates (4 mg wet tissue) in 0.05 mol/L phosphate buffer containing 1.15% (w/v) KCl was mixed in a quartz cuvette with 935 mL of the coupling solution prepared by dissolving 33.6 mg disodium EDTA, 6.5 mg NaN3, 30.7 mg of GSH, 16.7 mg NADPH and 100 units of GSH reductase in 100 mL of 50 mmol/L Tris-HCl (pH 7.6). Kinetic decay of NADPH fluorescence (Ex. 355 nm/Em. 465 nm) was measured after the addition of 25 mL of 1 mmol/L H₂O₂ as substrate using a Hitachi model 650-60 fluorescence spectrophotometer. The whole procedure was carried out as previously described[10].

SOD activity measurement

One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of epinephrine auto-oxidation to adrenochrome. For Mn SOD activity measurement 1 mmol/L KCN was added to the reaction mixture to inhibit CuZn SOD activity which was approximated by subtracting Mn SOD activity from total SOD activity as previously described[11]. Protein content was assayed by the Bradford method (Bio-Rad, Hercules, Calif., USA).

Determination of uninary 8-OHdG by competitive ELISA

Urine samples were centrifuged at 10 000 r/min for 15 min, and the supernatant was used for determination of 8-OHdG by competitive ELISA using the “8-OHdG Check” kit. The determination range was 0.64-2000 μg/ mL and the specificity of the monoclonal antibody used in the competitive ELISA kit was established.

Statistical analysis

Significance was tested by the paired t-test or analysis of variance. Results were presented as mean ± SD. P < 0.05 was considered statistically significant.

RESULTS

MDA and LPO formation in liver and kidney homogenate and plasma was considerably increased in the model group. However, in the mistletoe alkali-treated groups, no significant increase in MDA and LPO formation was observed (Table 1, Table 2 and Table 3). Results showed that hepatotoxicity and nephrotoxic nephritis in rats were induced by CCl₄ with formation of lipid peroxide. However, mistletoe alkali was effective in reducing malonyl dialdehyde (MDA) content in the tissues, and had good scavenging effects on hydroxyl free radicals.

Table 1 LPO, GSH- Px, SOD in rat liver (n = 32, mean ± SD).

Groups	Dosage	LPO	GSH-Px	SOD
	(mg/kg)	(nmoL/mg protein)	(nmoL/mg protein)	(nmoL/mg protein)
Normal control	-	11.142 ± 0.916b	18.713 ± 3. 587b	31.537 ± 2.848b
CCl4 + MA group	90	10.326 ± 1.213b	20.403 ± 2.388c	39.901 ± 2.667c
CCl4 + vit E group	400	11.221 ± 2.182b	16.353 ± 2.962b	27.736 ± 3.646b
CCl4 group	-	17.271 ± 1.108	13.342 ± 3.636	21.639 ± 3.991

^bP < 0.01, ^cP < 0.05 vs CCl₄ group.

Table 2 LPO, GSH- Px, SOD in rat kidney (n = 32, mean ± SD).

Groups	dosage	LPO	GSH-Px	SOD
	( mg/kg)	(nmol/mgprotein)	(μ/g mL)	(μ/mg protein)
Normal control	-	1.268 ± 0.99b	7.174 ± 0.576c	39.573 ± 7.322c
CCl4 + MA group	90	1.973 ± 0.512b	7.431 ± 0.483c	37.887 ± 6.979b
CCl4 + Vit E group	400	1.312 ± 0.418b	6.698 ± 0.746b	39.399 ± 8. 684c
CCl4 group	-	3.826 ± 0.289	4.576 ± 0.611	22.639 ± 5.342

^bP < 0.01,

^cP < 0.05 vs CCl₄ group.

Table 3 MDA, SOD, GSH- Px in blood plasma (n = 32, mean ± SD).

Groups	Dosage	MDA	GSH-Px	SOD
	(g/kg)	(mol/L)	(μ/mL)	(μ/mL)
Norma control	-	14.341 ± 2.01	21.17 ± 3.82b	19.01 ± 2.36b
CCl4 + MA group	90	12.88 ± 3.98b	22.67 ± 3.42b	23.62 ± 3.17c
CCl4 + Vit E group	400	14.91 ± 2.79	18.77 ± 1.98b	17.63 ± 1.88b
CCl4 group	-	14.98 ± 3.63	16.11 ± 2.41	14.88 ± 3.15

^bP < 0.01,

^cP < 0.05 vs CCl₄ group.

The effective protection of liver and kidney against oxidative damage induced by mistletoe alkali was further demonstrated by determining GP_X and SOD activity in the Liver and kidney homogenate and plasma. GP_X and SOD activity decreased markedly in the liver and kidney homogenate and plasma in model group, but activity loss was prevented by mistletoe alkali in the pretreatment groups. GP_X and SOD activity increased slightly compared to normal group. The data in the present study suggested that mistletoe alkali could be a potential herbal medicine for improving GP_X and SOD activity in liver and kidney tissue and plasma. The enzymes level is shown in Table 1, Table 2 and Table 3. The scavenging activity was steadily increased with the increase of drug density, suggesting that the free radical scavenging activity was related with mistletoe alkali density.

The DMPO-OH signal produced by the Fenton reaction was reduced by the addition of mistletoe alkali in a concentration dependent manner (Table 4). The 50% inhibitory concentration of mistletoe alkali for DMPOI-OH formation was compared to that for Trolox as a reference radical scavenger. The results indicated that mistletoe alkali not only scavenged · OH directly but also inhibited · OH generation in the Fenton reaction. This was further confirmed when the · OH scavenging activity of mistletoe alkali was determined in the H₂O₂/UV system as · OH source. In this system, the Trolox equivalence of the mistletoe alkali solution was about 0.1 mmoL/L (data not shown).

Table 4 Effects of mistletoe alkali on Fenton and H₂O₂/UV-mediated DMPO-OH formation.

Drug concentration (μg/ mL)	Sample size (n)	Opposite high peak of ESR (mean ± SD)	Clearance rate (%)
0	20	38.71 ± 3.62	_
2	20	34.16 ± 3.75	11.67
10	20	29.42 ± 2.99	47.82
50	20	22.65 ± 4.83	67.67
100	20	14.38 ± 2.76	91.28

There was a significant difference in urinary excretion of 8-OHdG between the control group and 900 mg/kg mistletoe alkali-fed group (7.84 ± 3.72, 3.26 ± 1.92 μlmoL/d, P < 0.05).

DISCUSSION

There is a growing body of evidence that oxygen-derived free radicals are involved in the pathogenesis of over 50 human diseases[12]. Antioxidant therapy aimed at reducing free radical-mediated tissue damage represents a rational approach in preventing the onset and/or progression of free radical-related tissue damage. In this connection, the measurement of antioxidant activity should form an additional basis for drug screening and selection[13]. In the present study, we examined the antioxidant potential of a traditional Chinese medicine, Viscum coloratum (Komar.) Nakai.

Recently, rapid progress has been made in research of Viscum coloratum (Komar.) Nakai. The preparation of Oujisheng has already been applied extensively in treatment of cancer[14]. China has a long history in research of Viscum coloratum (Komar.) Nakai. In 1934, Jingli Tong discovered that the Chinese Viscum coloratum (Komar.) Nakai (Huangguo Viscum coloratum (Komar.) Nakai) can lower blood pressure[15].

The involvement of free radical-mediated oxidative process in the development of CCl₄ hepatotoxicity is well established[16]. It was reported that impairment of hepatic antioxidant status is associated with a substantial hepatocellular damage induced by CCl₄[17]. GSH is a crucial determinant of tissue susceptibility to oxidative damage, and the depletion of hepatic GSH content has been shown to be associated with an enhanced toxicity to chemicals including CCl₄[18]. The liver protection afforded by pretreatment with NAC, a precursor for GSH synthesis, and vitamin E, an inhibitor of lipid peroxidation, indicates that both the sustained hepatic GSH content and the lipid peroxidation inhibition are important factors involved in protecting against CCl₄ hepatotoxicity. Changes in the sensitivity of tissue homogenates to in vitro oxidative challenge have been used as an indicator of altered tissue susceptibility to free radical-induced oxidative injury in vivo[19]. Our results, indicating an increase in sensitivity of tissue homogenates to ferric ion-induced lipid peroxidation following CCl₄ treatment, have validated the application of “forced peroxidation” assays in measuring alterations in tissue after oxidative challenge.

To our knowledge, there is no report indicating that the use of MA in vivo studies counteracts CCl₄ toxicity. Mistletoe alkali pretreatment at a daily dose of 900

mg/kg body weight for 6 d significantly enhanced hepatic antioxidant status in CCl₄-treated rats, with a concomitant reduction of hepatocellular damage, suggesting that the involvement of GSH-enhancing action can protect liver against damage.

8-OHdG, a DNA base-modified product generated by reactive oxygen species, is mutation prone and a good marker for oxidative damage. It has been hypothesized that oxidative damage can occur in DNA during the peroxidative breakdown of membrane polyunsaturated fatty acids. Lipid peroxidation can mediate 8-OHdG formation in vitro and may play a role in carcinogenesis by inducing 8-OHdG generation in vitro. In this study, the amount of 8-OHdG in the urine of rats fed with mistletoe alkali was significantly lower than that in the control rats, suggesting that mistletoe alkali plays a role in the prevention of oxidative DNA damage in vivo.

In conclusion, increased level of free radicals is associ-ated with decreased antioxidant status in liver and kidney of rats treated with CCl₄. This finding is similar with other investigations[20,21]. Mistletoe alkali strongly inhibits lipid peroxidation and thereby decreases liver and kidney damage.