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Oral Administration of Lycopene Reverses. Cadmium-suppressed Body Weight Loss and Lipid. Peroxidation in Rats. Nadir Rencuzogullari & Suat Erdogan.
Biol Trace Elem Res (2007) 118:175–183 DOI 10.1007/s12011-007-0027-7

Oral Administration of Lycopene Reverses Cadmium-suppressed Body Weight Loss and Lipid Peroxidation in Rats Nadir Rencuzogullari & Suat Erdogan

Received: 20 February 2007 / Accepted: 12 March 2007 / Published online: 8 May 2007 # Humana Press Inc. 2007

Abstract Cadmium (Cd) exposure has been recognized to result in a wide variety of cellular responses, including oxidative stress and body weight loss. The aim of the present study was to examine the effect of lycopene supplementation on the antioxidant defense system, lipid peroxidation (LPO) level, nitric oxide (NO), tumor necrosis factor alpha (TNF-α) production, and body weight in Cd-exposed rats. Animals were divided into four groups (n=7): control, Cd-treated, Cd plus lycopene-treated, and lycopene-treated. Cadmium (as CdCl2) was administrated orally for 20 days (6.6 mg kg−1 day−1), and lycopene (10 mg kg−1 day−1) was similarly administered. Lycopene administration significantly suppressed Cd-induced LPO in plasma and kidney homogenates. Lycopene also reversed Cd-decreased body weight compared to the control. Cadmium treatment had diverse effects on the antioxidant enzyme activities. Although antioxidant superoxide dismutase activity was unchanged, glutathione peroxidase activity was decreased, and catalase activity was elevated in kidney homogenates of Cd-administrated group. However, lycopene treatment reversed Cd-changed enzyme activities to the control level. Xanthine oxidase activity and TNF-α concentration were not altered by Cd administration, indicating that superoxide anion production and inflammation were not stimulated. Cadmium did not change NO levels in kidney homogenates but decreased those in plasma, and this effect was not prevented by lycopene supplementation. The result suggests that consumption of adequate levels of lycopene may be useful to prevent heavy-metal-induced LPO and body weight loss.

Keywords Antioxidant . Cadmium . Lipid peroxidation . Lycopene . Oxidative stress

N. Rencuzogullari : S. Erdogan (*) Department of Biochemistry, Faculty of Veterinary Medicine, Mustafa Kemal University, Tayfur Sokmen Campus, Antakya, Hatay 31040, Turkey e-mail: [email protected]

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Introduction Cadmium (Cd) is a harmful industrial and environmental pollutant, which is found in air, drinking water, plants, and animal products. The flow of Cd in ecological systems increases through major sources as mining, smelting, and industrial use. Resources of human exposure to this metal include food, cigarette smoke, and beverages [1]. The molecular mechanisms of Cd toxicity are not yet well defined. In contrast to transition metals, the oxidative effect of Cd is indirect and based mainly on the depletion of SH-group-containing compounds [2]. Cadmium itself is unable to generate free radicals directly; however, indirect generation of various radicals involving the superoxide radical and nitric oxide (NO) has been reported [3]. Some experiments also confirmed the generation of hydrogen peroxide (H2O2), which itself in turn may be a significant source of radicals via Fenton chemistry [4]. Recent studies on mammals have shown that Cd stimulates formation of reactive oxygen species, including oxygen-free anion radical, H2O2, and probably hydroxyl radicals [5–7]. As a consequence, enhanced lipid peroxidation (LPO), DNA damage, altered calcium and sulfhydryl homeostasis, and marked disturbances to antioxidant defense systems occur [8, 9]. Cadmium can cause the development and progression of diabetic renal complications, hypertension, osteoporosis, leukemia, and cancer in several organs [10]. Lycopene is an antioxidative beta-carotene found in vegetables and fruit; tomatoes contain particularly high concentrations of lycopene [11]. Various epidemiology studies have provided evidence to indicate that diets containing high concentrations of carotenoids may lower risks for the development of chronic diseases such as cancer and heart disorders [12, 13]. Lycopene has received particular attention in recent years because of its highly efficient antioxidant and free radical scavenging capacity [11, 14]. Lycopene in the triplet state can return to the ground state by dissipating the energy as heat or by physical quenching, leaving the lycopene molecule intact and ready for further quenching events [15, 16]. Cantrell et al. [17] demonstrated that lycopene is also an excellent high singlet oxygen (O2) quencher in biological membrane models such as liposomes. In contrast, trapping of other reactive oxygen species, like hydroxyl radical (OH−), nitric oxide (NO2), or peroxynitrite, leads to oxidative breakdown of the lycopene molecule [18]. In organic solution, lycopene was the most rapidly destroyed carotenoid upon reaction with peroxyl radicals [19], indicating its presence in the first line of defense. Given this potent antioxidant function in vitro, lycopene may also protect in vivo against oxidation of lipids, proteins, and DNA [20]. There has been no model exploring the effect of lycopene on Cd exposure; therefore, we investigated the effects of lycopene on Cd toxicity concentrating on the antioxidant defense system, LPO, inflammation, and changes to body weight.

Materials and Methods Experimental Design and Management Twenty-eight adult male Wistar-Albino rats (∼175 g) were used in this study. The rats were obtained from a colony maintained under SPF conditions at the Faculty of Medicine, Experimental Research Laboratory, Firat University, Elazig, Turkey. Animals were randomly housed on the basis of weight in electrically heated batteries under fluorescent lighting and allowed ad libitum access to feed and water. After a period of 2 weeks of acclimatization, animals were divided into four groups (comprising seven each). The groups were (1) control, (2) Cd-treated, (3) Cd + lycopene-treated, and (4) lycopene-treated. Cadmium

Effects of Lycopene Supplementation on Cd Exposure

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was given orally as CdCl2 at a dose of 6.6 mg kg body weight−1 day−1, 1/15 LD50, whereas group Cd + lycopene was treated orally with the same amount of CdCl2 plus lycopene (10 mg kg body weight−1 day−1). Animals in the lycopene group were treated orally with 10 mg lycopene/kg body weight on a daily basis. Rats were treated by gavage every day with or without lycopene and/or CdCl2 for 20 days. The LD50 of CdCl2 when given orally to rats was reported to be 63–88 mg/kg body weight [21]. At the beginning and end of the experimental period body weight of rats were recorded. Animals were killed under anesthesia and kidney tissues were quickly excised, rinsed in ice-cold saline, and stored frozen at −20°C until analyzed. Blood samples were collected in heparinized tubes and placed immediately on ice. Plasma samples were obtained by centrifugation at 3,000 rpm for 5 min and stored at −20°C. Tissue Preparation Kidneys were removed immediately after they were killed and washed using chilled saline solution. Tissues were minced and homogenized separately in ice-cold potassium phosphate buffer (pH 7.4) in a Dunce type homogenizer. The homogenate was centrifuged at 15,000 rpm for 10 min at 4°C, and resultant supernatant was used for enzyme assays. Total protein concentration in kidney supernatants was assayed by the method of Bradford [22] using bovine serum albumin as a standard. Biochemical Assays Thiobarbituric acid-reactive substances (TBARS) were determined in plasma and kidney homogenates using the spectrophotometric method described by Yoshoiko et al. [23]. The values of TBARS material were expressed in terms of malondialdehyde (MDA) concentration (μMol/l). Because NO measurement is very difficult in biological specimens, tissue nitrite (NO2 ) and nitrate (NO3 ) were estimated as an index of NO production. The method for plasma and tissue total nitrite (nitrite and nitrate) levels was based on the Griess reaction [24]. Total nitrite (nitrite and nitrate) was measured using a spectrophotometer at 545 nm after conversion of nitrate to nitrite by copperized Cd granules. Total superoxide dismutase (SOD) activity was determined in kidney tissue samples according to the method of Sun et al. [25]. The principle of the method is based on the inhibition of nitroblue tetrazolium reduction by the xanthine/xanthine oxidase (XO) system as a superoxide generator. Catalase (CAT) activity was assayed in kidney tissue according to the method of Aebi [26]. The method is based on the rate of H2O2 degradation by the action of CAT contained within the test samples and is followed by spectrophotometric measurement at 240 nm. Glutathione peroxidase (GSH-Px) activity was measured in tissue homogenates by a kinetic method using a commercial kit (RANSOD by Randox Lab., UK). The reaction is based on the measurement of NADPH consumption (i.e., NADPH oxidation by glutathione reductase) at 340 nm. Xanthine oxidase activity in kidney tissue was assayed spectrophotometrically at 293 nm and 37°C with xanthine as substrate [27]. The formation of uric acid from xanthine results in an increase in absorbency. One unit of activity was defined as 1 μmol of uric acid formed per minute at 37°C, pH 7.5. Tissue samples were treated with nitric acid and hydrochloric acid digestion according to the methods of Rahel-Khazen et al. [28]. Cadmium and zinc (Zn) concentrations in kidney

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homogenates were analyzed by inductively coupled plasma–atomic emission spectrometry (ICP-AES; Liberty Series-II Varian, USA) at 228.802 and 213.856 nm, respectively. Plasma tumor necrosis factor-alpha (TNF-α)? levels were determined using the ELISA rat TNF-α kit (Diaclone, France). All samples were assayed in triplicate. Statistical Analysis All statistical analyses were subjected to one-way analysis of variance by using SPSS statistical analyses system configured for the computer (SPSS for Windows version 9.05). If appropriate, post hoc analyses were carried out using Duncan’s test. All statements of significance are based on the 0.05 level of probability. All data are presented as means ± SE.

Results and Discussion Effects of Cadmium and Lycopene on Body Weight Rats received Cd and/or lycopene once daily for 20 days as explained above. Cadmium treatment decreased body weight (15.5%) at the end of study, although the change was not statistically significant. This result was comparable to those previously found in studies of chicken [29] and rats [30]. Cadmium-reduced body weight was reversed (∼9%) by lycopene administration, although the effect was not complete. We have previously demonstrated that the administration of antioxidants such as ascorbic acid has a partial preventative effect on Cd-decreased body weight loss in chicken [29]. Similar to that study, administration of lycopene was shown to reduce Cd-depressed body weight in the present work. The adverse effect of Cd on body weight was presumably related to its toxicity on many body systems and the mechanism may involve the induction of free radical production [31]. Cadmium toxicity has previously been shown to result in a wide variety of cellular responses, including oxidative stress and LPO especially in liver and kidney. Any reversal in body weight by lycopene supplementation may be because of the antioxidant properties associated with this carotenoid molecule.

Lipid Peroxidation and Antioxidant System Cadmium is known to induce the production of free radicals such as OH−, NO2 and superoxide anion (O2 ) [31]. However, the exact mechanism by which Cd induces oxidative stress remains to be clarified. Malondialdehyde is an end product of LPO, thus the level of MDA can be used to estimate the degree of LPO. Lipid peroxidation in plasma and kidney homogenates was determined by increased TBARS formation in rats treated with Cd. As shown in Fig. 1, MDA concentrations in plasma and kidney homogenates of rats exposed to Cd was significantly increased compared to the control. The supplementation of lycopene significantly reversed MDA levels in plasma (p