Protective effects of blueberries (Vaccinium corymbosum L.) extract against cadmium-induced hepatotoxicity in mice

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.elsevier.com/locate/etap

Protective effects of blueberries (Vaccinium corymbosum L.) extract against cadmium-induced hepatotoxicity in mice Pin Gong a,∗ , Fu-xin Chen b , Lan Wang a , Jing Wang a , Sai Jin a , Yang-min Ma a a

College of Life Science and Technology, College of Chemistry and Chemical Engineering, Shaanxi University of Science and Technology, Xi’an 710021, China b School of Chemistry and Chemical Engineering, Xi’an University of Science and Technology, Xi’an 710054, China

a r t i c l e

i n f o

a b s t r a c t

Article history:

The oxidative status and morphological changes of mouse liver exposed to cadmium chlo-

Received 26 November 2013

ride (Cd(II)) and therapeutic potential of blueberry (Vaccinium corymbosum L.) extract against

Received in revised form

Cd(II)-induced hepatic injury were investigated. A variety of parameters were evaluated,

18 March 2014

including lipid peroxidation (LPO), protein carbonyl (PCO) level, DNA fragment, as well as

Accepted 23 March 2014

antioxidative defense system (i.e., superoxide dismutase (SOD), catalase (CAT), reduced glu-

Available online 30 March 2014

tathione (GSH)). Elemental analysis and evaluation of morphological changes and NO levels were also performed. Exposure to Cd(II) led to increased LPO and PCO as well as DNA frag-

Keywords:

ment and a reduction of SOD and CAT activities, however, the content of GSH elevated

Blueberry

probably due to biological adaptive-response. In contrast, co-treatment of anthocyanin (Ay)

Anthocyanin

inhibited the increased oxidative parameters as well as restored the activities of antiox-

Cadmium

idative defense system in a dose-dependent manner. Ay administration regained these

Hepatotoxicity

morphological changes caused by intoxication of Cd(II) to nearly normal levels. Moreover,

Oxidative stress

the accumulation of Cd(II) in liver may be one of the reasons for Cd(II) toxicity and Ay can chelate with Cd(II) to reduce Cd(II) burden. The influence of Cd(II) on the Zn and Ca levels can also be adjusted by the co-administration of Ay. Exposure to Cd(II) led to an increase of NO and Ay reduced NO contents probably by directly scavenging. Potential mechanisms for the protective effect of Ay have been proposed, including its anti-oxidative and antiinflammatory effect along with the metal-chelating capacity. These results suggest that blueberry extract may be valuable as a therapeutic agent in combating Cd(II)-induced tissue injury. © 2014 Elsevier B.V. All rights reserved.

Abbreviations: Ay, anthocyanins; BHT, butylated hydroxytoluene; Ca, calcium; CAT, catalase; Cd(II), cadmium; Cd(II), cadmium chloride; DNPH, 2,4-dinitrophenylhydrazine; DTNB, 5,5 -dithiobis(2-nitrobenzoic acid); GSH, reduced glutathione; i.g., intragastric; i.p., intraperitoneal; LPO, lipid peroxidation; NO, nitric oxide; PBS, phosphate buffer solution; PCO, protein carbonyl; ROS, reactive oxygen species; SOD, superoxide dismutase; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive; TCA, trichloroacetic acid; Zn, zinc. ∗ Corresponding author at: College of Life Science and Technology, Shaanxi University of Science and Technology, Xi’an 710021, China. Tel.: +86 29 86132711; fax: +86 29 86132711. E-mail address: [email protected] (P. Gong). http://dx.doi.org/10.1016/j.etap.2014.03.017 1382-6689/© 2014 Elsevier B.V. All rights reserved.

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1.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

Introduction

Exposure to toxic metals has become an increasingly recognized source of illness worldwide. Cadmium is the common environmental heavy metal pollutants and have widespread distribution. Besides occupational exposure, environmental exposure of Cd(II) occurs via diet, drinking water and through inhalation. It has been established that excess cadmium exposure produces adverse health effects on human being and contributes to a well-defined spectrum of diseases (Fowler, 2009; Jarup and Åkesson, 2009; Matés et al., 2010). Acute Cd(II) intoxication primarily results in liver accumulation and hepatocellular damage (Arroyo et al., 2012). Although several mechanisms have been proposed to explain the Cd(II)-induced hepatic toxicity, no mechanisms have been yet defined explicitly. Epidemiological and experimental evidences suggested that acute Cd(II)-induced liver injury is considered a biphasic process including an initial phase caused by direct metal actions and ischemia, and a latter one due to inflammation and oxidative stress (Tzirogiannis et al., 2003; Cuypers et al., 2010). Recently, various studies are focused on the development of suitable reagents to counteract the toxic effect of Cd(II). Several antioxidants and metal-chelating agents were proved effective in protecting against Cd(II)-induced hepatotoxicity (Nemmiche et al., 2007; Newairy et al., 2007; Borges et al., 2008; El-Sokkary et al., 2010). Our group has demonstrated that endomorphin 1 and caffeic acid phenethyl ester were potential agents to block the toxicity of Cd(II) (Gong et al., 2008, 2012). Although a lot of possible treatment protocols for Cd(II) intoxication have been investigated, only a few have been tested in clinical trials, such as zinc supplements (NCT00376987). Therefore, novel therapeutic agents with improved efficacy are needed to ameliorate or counteract the intoxication of Cd(II). Blueberries (Vaccinium corymbosum L.) have been shown to provide protection against oxidative stress, inflammation, carcinogenesis and chronic diseases (Graf et al., 2005; Lau et al., 2005; Mcdougall et al., 2008; Schmidt et al., 2006). As with other fruits, blueberries contain a high level of vitamin C (ascorbic acid), folic acid, resveratrol, pterostilbene and piceatannol (Rimando et al., 2004). However, blueberries are recognized as a good source of anthocyanins (Ay) (212 mg/100 g of fresh weight) that contribute to their beneficial effects on oxidative stress (Neto, 2007). Delphinidin, petunidin and malvidin are the major contributors to total anthocyanin contents (Lohachoompol et al., 2008). Ay, a class of naturally presenting polyphenol compounds, are water-soluble glycosides of polyhydroxyl and polymethoxyl derivatives of 2-phenylbenzopyrylium. Ay exist at low pH as a flavylium cation, which is the naturally occurring form. The flavylium cation is highly electron deficient, which leads to their potent activity toward free radicals and oxygen reactive species. Ay are known as a unique group of substances which are believed to provide a broad variety of health benefits such as prevention of heart disease, inhibition of carcinogenesis, anti-obesity (Bagchi et al., 2004; Galli et al., 2006; Tsuda et al., 2003) and benefit effect on eye health (Yao et al., 2010). Ay also possess powerful antioxidant (Shih et al., 2007), anti-inflammatory (Karlsen et al., 2007), and anti-tumor

properties (Shih et al., 2005). Moreover, Ay from black raspberries, blackberries, and strawberries exhibit protective effects against a number of hepatotoxic agents (Reen et al., 2006; Choi et al., 2009). Given the facts that oxidative stress and inflammatory process are critical mediators for Cd(II) intoxication progress, we hypothesized Ay may elicit hepatoprotective and antioxidant effects against the intoxication of Cd(II). To test this hypothesis, we examine the protective effects of Ay extracted from blueberry against Cd(II)-induced mice hepatic damage. According to Barros et al. (2006), animals ingested approximately 0.3–3.2 mg/kg/day Ay; thus, their dietary intake was approximately of the same order of magnitude as that which occurs in humans. As a result, in this experiment, we employ a dose of 0.3–30 mg/kg/day Ay to test the effect to protect against the toxicology of Cd(II). As blueberries have been consumed by the people all over the world, as one of their dietary items and there are no reported side effects on normal people, the results of the present studies may have future therapeutic relevance in the areas where humans are exposed to Cd(II) either occupationally or environmentally.

2.

Materials and methods

2.1.

Chemicals

Cadmium chloride, reduced glutathione (GSH), 5,5 acid) (DTNB), thiobarbituric dithiobis(2-nitrobenzoic acid (TBA), butylated hydroxytoluene (BHT) and 2,4dinitrophenylhydrazine (DNPH) were obtained from Sigma–Aldrich Chemical Company (St. Louis, MO, USA). Blueberries were collected in July 2010, in Ni Shaan town, Shaanxi province. Ay were prepared in our laboratory following the methods as described by Barnes et al. (2009). All other chemicals were of analytical grade and obtained from standard commercial supplies.

2.2.

LC–MS/MS analysis

The major constituents of Ay from blueberries were evaluated using liquid chromatography–mass spectrometer (LC–MS) methods (Lohachoompol et al., 2008). An EVOQ Qube LC–QQQ mass spectrometer fitted with an ESI interface (Bruker Corporation, USA) and coupled to a HPLC and PDA detector were employed. The analytical column employed was a ZORBAX Eclipse XDB-C18 USP L1 (Agilent USA, 250 mm length × 4.6 mm i.d.), was installed before the analytical column. The temperature of the column oven was maintained at 35 ◦ C. Mobile phase consisted of 3% formic acid (A) and acetonitrile (B). Ay were separated with the following stepwise gradient: 0–20 min, 20–100% B; 20–35 min, 100–20% B; 35–40 min, 20% B; the flow rate of the mobile phase was 0. 50 mL/min. The injection volume was 10 ␮L. The mass spectrometer was operated in the positive ion mode (ESI+) with the following operating parameters: capillary voltage 4.5 kV, end plate offset voltage −500 V, collision cell RF 200 Vpp. The source temperature was 150 ◦ C and the dry heater temperature was 180 ◦ C. Dry gas flow was 8 L/min.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

2.3.

Animals

2.6. Estimation of lipid peroxidation and protein carbonyl content

Thirty-five healthy Kunming male mice (SPF grade) with body weight between 18 and 20 g were obtained from the animal center of the Forth Military Medical University. The animals were kept in an environment with controlled temperature (24–26 ◦ C), constant humidity (55–60%) and controlled photoperiod (12 h of light and 12 h of dark) properties for 3–5 days week before the start of experiment. A commercially balanced diet and tap water were provided ad libitum. All animals were cared for and experiments were performed out in accordance with the European Community guidelines for the use of experimental animals (86/609/EEC). All the protocols in this study were approved by the Ethics Committee of Shaanxi University of Science and Technology, China.

2.4.

Experimental protocol

CdCl2 and Ay were, respectively, dissolved in 0.9% saline, then diluted to required concentration immediately before use. Mice were randomly divided into five groups (7 mice/group). As indicated below, treatment groups consisted of control, damage, and the three different doses of Ay administered groups. Groups I

Control

II

Cd(II)

III

Cd(II) + Ay0.3

IV

Cd(II) + Ay3

V

Cd(II) + Ay30

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Treatment Saline solution solution i.p. Saline solution (2 mg/kg/day) i.p. Ay (0.3 mg/kg/day) (2 mg/kg/day) i.p. Ay (3 mg/kg/day) (2 mg/kg/day) i.p. Ay (30 mg/kg/day) (2 mg/kg/day) i.p.

i.g. + saline i.g. + CdCl2 i.g. + CdCl2 i.g. + CdCl2 i.g. + CdCl2

Mice in groups III–V were co-treated daily with Ay through intragastric (i.g.) administration at different doses, groups I and II were given the same volume of saline solution, consecutively 14 days. The toxin-treated animals were administered continually intraperitoneal (i.p.) injections of CdCl2 (0.1 mL) at dose of 2 mg/kg bw/day for 14 days till sacrifice. Control group was received the same volume of saline. At the end of experimental period, i.e. after 14 days, the mice were deprived of food overnight, and sacrificed by decapitation. Mice were immediately subjected to necropsy and liver tissues were promptly isolated, cleaned from of adhering matters, washed with saline solution, and stored at −70 ◦ C for the biochemical studies.

2.5. Determination of plasma transaminases (AST and ALT) activities Plasma enzymes AST and ALT were used as the biochemical markers for the early hepatic damage (Reitman and Frankel, 1957), and were determined using a commercial Kit (Jiancheng, Nan Jing, China).

Lipid peroxidation in liver was estimated spectrophotometrically by measuring thiobarbituric acid reactive substances by the method of Niehius and Samuelson (1968). In brief, 0.5 mL of tissue homogenate was treated with 2 mL of TBA–trichloroacetic acid (TCA)–HCl reagent (0.37% TBA, 15% TCA, 0.25 M HCl, 1:1:1 ratio) and placed for 30 min in a boiling water bath, then cooled and centrifuged for 10 min at room temperature and the supernatant was measured at 535 nm using a blank containing all the reagents except the sample. MDA content of the sample was calculated using the extinction co-efficient of MDA, which is 1.56 × 105 M−1 cm−1 . As a hall mark of protein oxidation, total protein carbonyl content was determined in the livers by a spectrophotometric method described by Levine et al. (1999). In brief, tissue homogenate was centrifuged at 10,000 × g for 20 min to separate cytosol, and then 0.5 mL of cytosolic fraction, 0.5 mL of TCA were added. Later, 0.5 mL of DNPH was added and kept for1 h in room temperature. Pellet was washed thrice with 1 mL of ethanol–ethylacetate mixture, and then the pellet was dissolved in 1 mL of guanidinehydrochloride, the developed color was read at 365 nm. Results were expressed as nmol of DNPH incorporated/mg protein based on the molar extinction coefficient of 22,000 M−1 cm−1 for aliphatic hydrazones.

2.7.

Assay of DNA fragmentation

The DNA damage in hepatic tissue as a result of Cd(II) exposure and its protection by Ay was determined following a DNA fragmentation assay as described by Lin et al. (1997). Briefly, hepatic tissue homogenates were treated with 100 mM Tris–HCl buffer, pH 8.0, 1 mM EDTA and 0.5% triton X-100 and centrifuged. The supernatant was transferred carefully in a tube and 1 mL of 25% TCA was added to it, the mixture were vortex vigorously and incubated overnight at 4 ◦ C. Quantitative analysis of DNA was carried out by diphenylamine reaction. The percentage of fragmentation was calculated from the ratio of DNA in supernatant to the total DNA. The extent of DNA fragmentation has also been assayed by electrophoresing genomic DNA samples, isolated from normal as well as experimental mouse livers, on agarose/ethidium bromide gel (Sellins and Cohen, 1987).

2.8.

Determination of non-enzymatic antioxidant

Reduced glutathione (GSH) was determined by the method of Moron et al. (1979). To 1 mL of supernatant treated with 0.5 mL of Ellman’s reagent (19.8 mg of 5,5 -dithiobisnitro benzoic acid (DTNB) in 100 mL of 0.1% sodium citrate) and 2 mL of phosphate buffer (0.2 M, pH8.0) and 0.5 mL of DTNB were added. The absorbance was read at 412 nm. To prevent the autooxidation of GSH, the samples were reduced with potassium borohydride prior to analysis.

2.9.

Assay of activities of antioxidant enzymes

Superoxide dismutase (SOD) activity was determined from its ability to inhibit the autoxidation of pyrogallol using a

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e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

modification of the method of Marklund and Marklund (1974). Briefly, samples were assayed in a solution of 3 mL of 50 mM phosphate buffer (pH 8.2) and 10 ␮L of 50 mM pyrogallol (in 10 mM HCl). The rate of pyrogallol auto-oxidation was measured at 325 nm at 25 ◦ C. One unit of enzyme activity was defined as the amount of the enzyme, which gave 50% inhibition of the auto-oxidation rate of 0.1 mM pyrogallol in 1 mL of solution at 25 ◦ C. Catalase (CAT) assay was adopted from the method of Aebi (1984), based on the principle that at ultraviolet range H2 O2 shows a continual increase in absorption with decreasing wavelength. The rate of H2 O2 decomposition was followed by monitoring the absorbance at 240 nm. In this assay, 250 ␮L of 0.66 M H2 O2 , 200 ␮L of 0.5 M phosphate buffer and 50 ␮L of tissue homogenate were added. Control contained only H2 O2 and phosphate buffer. The optical density was measured at 240 nm for 30 s. One unit of catalase activity is defined as the amount of enzyme required to decompose 1 ␮mol of hydrogen peroxide in 1 min. The activity was calculated using the extinction coefficient of H2 O2 (ε = 0.0394 mM−1 cm−1 ).

2.10.

Determination of protein content

The protein contents of the experimental samples were measured by the method of Bradford (1976) using crystalline BSA as standard.

2.11.

Histological studies

For qualitative analysis of hepatic histology, liver tissues were first fixed in 10% buffered neutral formalin solution for 72 h, and then immediately dehydrated in graded series of ethanol, immersed in xylol and embedded in paraffin. Sections of 4–5 ␮m were mounted. After deparaffinized, the sections were rehydrated, stained with hematoxylin and eosin, and subsequently subject to pathological assessment using an Olympus BX 51 Microscope. Photographs representative of the pathology were taken using an Olympus Camedia C-3040ZOOM digital camera.

2.12.

standing at room temperature for 10 min. After the mixture had reached equilibrium, the absorbance of the solution was then measured spectrophotometrically at 510 nm in a Hitachi model 557 UV spectrometer (Hitachi High Technologies, Japan). All tests and analyses were run in triplicate and averaged. The percentage of inhibition of Fe2+ –1,10phenanthroline monohydrate complex formation was given below: % Inhibition =

1−

A1 A0



× 100

where A0 was the absorbance of the control, and the A1 was the absorbance in the presence of the sample of Ay. The control did not contain (NH4 )2 Fe(SO4 )2 and 1,10-phenanthroline monohydrate, complex formation molecules.

2.14.

Nitric oxide (NO) content determination

As 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 nitrite and nitrate levels was based on the Griess reaction. All procedures were performed at 4 ◦ C. Liver samples were homogenized in ten times the tissue volume of ice-cold Tris–HCl buffer (50 mM, pH 7.4). After homogenization, samples were deproteinized with 75 mM ZnSO4 and 55 mM NaOH, and supernatants were used. One aliquot of supernatant was used for nitrite assessment by diazotization of sulfanilamide and coupling to naphthylethylene diamine. Another aliquot of supernatant was taken for the determination of total nitrite and nitrate levels which were reduced by copper-coated Cd granules in glycine buffer at pH 9.7 and then by diazotization of sulfanilamide and coupling to naphthylethylene diamine. Absorbance of the colored reaction product was measured at 545 nm. Nitrate levels were taken as differences between absorbance values of two aliquots. A standard curve was obtained with solutions containing 2–10 mM sodium nitrate. Data in this study presents the sum of nitrite and nitrate levels, which are NO metabolites and expressed as nmol/mg protein.

Determination of essential metal concentration 2.15.

The liver tissues were weighed and dry-ashed in a muffle furnace. The ash was solubilized with 3 M HNO3 and appropriately diluted. Samples were analyzed for Cd(II) (228.8 nm), Zn (213.9 nm), and Ca (422.7 nm) using a polarized Zeman Atomic Absorption spectrometer (Hitachi Z-200, Hitachi, Tokyo, Japan). The element contents are expressed as micrograms of the element per gram of wet tissue weight (␮g/g w.t.w.) (Brzóska et al., 2002; Jurczuk et al., 2003).

2.13.



Metal chelating assay

The chelation of ferrous ions by Ay was estimated by the method of Dinis et al. (1994) and Gülcin et al. (2004) with slight modifications. Briefly, different doses of Ay (1 mg/mL) were added to a solution of 1 mM (NH4 )2 Fe(SO4 )2 in NaOAc/HOAc buffer (pH 4.5). The reaction was initiated by the addition of 4 mM 1,10-phenanthroline monohydrate in NaOAc/HOAc buffer (pH 4.5). The mixture was vigorously shaken and left

Statistical analysis

All the values are represented as mean ± S.E.M. (n = 7). The statistical differences among different groups were analyzed by one-way analysis of variance (ANOVA). p-Values of 0.05 or less were considered significant.

3.

Results

3.1.

Plasma AST and ALT activities

Exposure to Cd(II) induced a significant increase on plasma AST (p < 0.01) which were 2.4-times higher than those in Control group, treatment with different dose of Ay were efficient in restoring AST levels toward Control group. Similarly, the intoxication of Cd(II) also cause the elevated activity of ALT(p < 0.05), around 4.9-fold. Therapy with Ay was effective in restoring enzyme activity at Control group (Fig. 1).

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

Fig. 1 – Effect of Cd(II) intoxication and co-treatment with different dose of Ay on plasma AST and ALT activities in mice. Data are mean ± S.E.M.; n = 7 (**p < 0.01, *p < 0.05 vs. Control group and ### p < 0.001, ## p < 0.01, # p < 0.05 vs. Cd(II) group).

3.2. Identification of the major content of blueberries extract According to the LC–MS/MS data, the major components of Ay in blueberries extract are delphinidin, petunidin and malvidin with in accordance with those reported by Lohachoompol et al. (2008). The detected mass, percentage of total Ay, proposal structures were listed in Table 1.

3.3.

Estimation of MDA and PCO contents

As depicted in Fig. 1, the liver MDA levels in Cd(II) group significantly elevated in response to Cd(II) treatment compared with control (p < 0.01), indicated that the treatment of Cd(II) caused obviously oxidative damages on mice. We found the increase was dramatically diminished by co-treatment of Ay in a dose-dependent manner (Fig. 2A and B). PCO is also considered to be an important parameter of oxidative stress. The elevated levels of PCO have been observed in the Cd(II) intoxicated hepatic tissue of the experimental animals (Fig. 2B). Ay co-treatment was found to be effective in preventing the Cd(II)-induced alterations.

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Fig. 2 – The effects of Ay on hepatic oxidative parameters in Cd(II)-exposed mice. (A) LPO levels. (B) PCO levels. Results are expressed as mean ± S.E.M.; n = 7 (***p < 0.001, **p < 0.01 vs. Control group and ## p < 0.01, # p < 0.05 vs. Cd(II) group).

3.4.

Assessment of DNA fragmentation

Fig. 3 represents the extent of DNA fragmentation. In Fig. 3A, a smear on agarose gel has been observed in Cd(II)-treated group, indicating random DNA degradation. The combination use of Ay can prevent the formation of smear. Moreover, quantitative measurement of DNA fragmentation has been assayed. Data showed that the intoxication of Cd(II) can increase the extent of DNA fragmentation and could be ameliorated by the treatment of Ay in a dose-dependent way.

3.5.

Activities of non-enzymatic antioxidant

The contents of GSH were increased by 65.1% (p < 0.01, compared to Control group), after exposed to Cd(II) (2 mg/kg per day) for 14 days, which may relate to an adaptive response to the liver damage. The effects of Ay (30 mg/kg per day) can decrease the concentrations of GSH to 39.1% (p < 0.01 compared to Cd(II) group) (Fig. 4).

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3.6.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

Activities of antioxidant enzymes

The activities of the antioxidant enzymes, SOD and CAT in the liver tissue of the experimental animals have been shown

in Fig. 5. Current findings represent that Cd(II) administration significantly attenuated the activities of the antioxidant enzymes compared to normal. Results show that co-treatment with Ay was able to prevent Cd(II)-induced alternated

Table 1 – Major constituents extracted from blueberries. Number

Experimental m/z

Theoretical Formula m/z

% of total Ay

465.1067

465.102753

C21 H21 O12

35.4

Delphinidin-3-galactoside

2

493.1380

493.134053

C23 H25 O12

26.1

Malvidin-3glucoside/malvidin-3galactoside

3

463.1269

463.123488

C22 H23 O11

8.6

Peonidin-3glucoside/malvidin-3arabinoside

1

Species

Potential structure

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

1021

Table 1 – (Continued) Number

Experimental m/z

Theoretical Formula m/z

% of total Ay

4

610.1878

610.160661

29.9

C27 H30 O16

Species

Potential structure

Quercetin-3-rutinoside

Fig. 4 – The effects of different concentration of Ay on liver GSH levels in Cd(II)-exposed mice. Results are expressed as mean ± S.E.M.; n = 7 (**p < 0.01 vs. Control group and # p < 0.05, ## p < 0.01 vs. Cd(II) group).

Fig. 3 – (A) DNA fragmentation pattern of the Cd(II)-induced liver damage on agarose/EtBr gel. DNA isolated from experimental liver tissues was loaded onto 1.5% (w/v) agarose gels. Lane 1: DNA isolated from normal liver; Lane 2: DNA isolated from Cd(II) intoxicated liver; Lanes 3–5: DNA isolated from Ay treated testes samples. (B) Effect of Ay on the extent of DNA fragmentation in the liver tissue of the experimental mice. Con: normal mice, Cd(II): Cd(II) treated mice, Ay+ Cd(II): mice treated with different dose of Ay following Cd(II) administration. Each column represents mean ± S.E.M., n = 6. *p < 0.05: The significant difference between the vehicle control and toxin treated groups and # p < 0.05: the significant difference between the toxin treated and Ay treated groups.

activities of the antioxidant enzymes in a dose-dependent manner.

3.7.

Histological observation

Fig. 6 represents the histological findings of the liver tissue of normal and experimental group of mice. Liver histology of control mice showed normal hepatic cord pattern, hepatic lobules and hepatocytes (Fig. 6A). Cd(II) exposure caused severe pathological lesions in hepatic tissues, these lesions consisted of loss of the parenchymal architecture, apparent broad hepatocellular swelling and lysis of hepatocyte plasma membranes after Cd(II) challenge. Multifocal liver cells degeneration and zonal coagulative necrosis were also observed (Fig. 6B and C). These pathological alterations were dramatically ameliorated in the liver of mice with the co-treatment of Ay (Fig. 6D).

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group, p < 0.01), Ay (30 mg/kg per day) co-treatment can block the action of Cd(II) in decreasing the Zn level and an increase of Zn content was observed (to 1.66 folds of Cd(II)-treated group, p < 0.01). An increase was observed in the levels of Ca for a treatment by 2 mg/kg per day Cd(II) (to 2.9 folds compare to untreated animals (p < 0.05)), and the effect of Ay can restore the alteration of Ca levels (to 38.3% of Cd(II)-treated group, p < 0.05). To explore the potential mechanism of Ay on the altered concentration of metal, the metal chelating abilities of Ay were tested. As shown in Fig. 8, the fact that the formation of the Fe2+ –1,10-phenanthroline monohydrate complex is not complete in the presence of Ay indicated that Ay can chelate iron. The absorbance of Fe2+ –1,10-phenanthroline monohydrate complex was linearly decreased in a dose-dependent manner. The percentage of metal chelating capacity of 0.1 mg/mL Ay was found as 90.6%.

4.

Fig. 5 – The effects of different concentration of Ay on liver CAT (A) and SOD (B) activities in Cd(II)-exposed mice. Results are expressed as mean ± S.E.M.; n = 7 (***p < 0.01, *p < 0.05 vs. Control group and # <0.05, ## <0.01 vs. Cd(II) group).

3.8.

Measurement of NO content

A marked increase (to 2.13 folds of that in Control group, p < 0.001) was found in the levels of NO content in the liver of Cd(II)-treated group of mice, the co-treatment with Ay (30 mg/kg per day) can reduce the NO level to 70.4% (p < 0.01, compared to Cd(II) group) (Fig. 7).

3.9.

Elemental analysis and metal chelating

The data from Table 2 demonstrated the accumulation of Cd(II) in the liver of all groups except control group, 14 days after the administration of Cd(II). A significant elevated Cd(II) concentration was observed in the Cd(II)-treated group (to 258.9 folds of Control group; p < 0.001compared to Control group). This accumulation was reduced by co-treatment with Ay (30 mg/kg per day) and the content of Cd(II) in the liver was reduced to 41.5% compared to Cd(II) group (p < 0.01). The administration of Cd(II) resulted in a decrease of Zn levels in the livers of Cd(II)-treated group (to 57.2% of Control

Discussion

Cadmium represents a dangerous environmental and industrial pollutant. The accumulation of Cd(II) occurs unevenly in human tissues, liver and kidney is the most concentrated organs. In the current study, the administration of Cd(II) to mice lead to the accumulation of this metal in liver tissue versus those of controls, which is coincide with previous reports (El-Sokkary et al., 2010). Cd(II) accumulation in liver is a well-established event and has been recognized as an important mechanism of hepatic damage induced by this metal (Stohs and Bagchi, 1995). The fact that co-administration of Ay could reduce the levels of Cd(II) in liver promotes the investigation on the capacity of Ay on metal chelating, thus, it is to be taken into consideration that the capacity of Ay to chelate metal ions is one of potential mechanisms against hepatic damage by Cd(II). Exposure to Cd(II) lead to an increase in plasma ALT and AST activities, mainly due to the leakage of these enzymes from the liver cytosol into the blood stream, which can be used as an indication of cadmium hepatotoxic effect, which is coincided with Santos et al. (2005). Treatment of Ay was efficient to restore the activities of ALT and AST. An increasing number of evidences have shown the overproduction of reactive oxygen species (ROS) may be one of the causes for liver damage induced by Cd(II). Lipid peroxidation (LPO) is an index of oxidative stress and has been considered as the primary mechanism for Cd(II) toxicity. Despite of its disability for Cd(II) to generate free radicals directly, Cd(II) does elevate LPO in the liver soon after exposure, by displacing iron and copper from intracellular sites and sequently resulting in the initiation of Fenton reaction (Stohs and Bagchi, 1995). The formation of malondialdehyde (MDA) and 4-hydroxynonenal, the decomposition products of LPO, can further damage proteins and nucleic acid by chaotic cross linkage. PCO is also a reasonable marker of oxygen radical-mediated protein damage under various pathophysiological conditions (Gong et al., 2008). Increase of PCO in tissues has been implicated in Cd(II)-induced protein damage. In line with this, our results clearly indicate that treatment of animals with Cd(II) results in significant increase of LPO and PCO, demonstrating the

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Fig. 6 – (A–D) Representative photographs from the liver showing the protective effect of Ay on Cd(II)-induced oxidative hepatic injury in mice. (A) Hepatic tissue of a control mice, showing normal appearance (H&E, 40×). (B and C) Hepatic tissue of a Cd(II)-treated mice, showing hepatocyte necrosis (*) and inflammatory cell infiltration (→) (40×). (D) Hepatic tissue of a Ay (30 mg/kg per day) and Cd(II)-treated mice, showing moderate necrosis (*) (40×). Scale bar in A, 100 ␮m.

Table 2 – Effects of Ay on the trace elements concentrations of mouse liver induced by Cd(II) All element concentrations are expressed in g/g wet tissue, value represents mean ± S.D. for three mice. Group Control Cd(II) Cd(II) + Ay0.3 Cd(II) + Ay3 Cd(II) + Ay30 ∗ ∗∗ ∗∗∗ # ##

Fig. 7 – The effects of Ay on liver NO release when exposure to Cd(II). Results are expressed as mean ± S.E.M.; n = 7 animals per group (***p < 0.001 vs. Control group and ## <0.01 vs. Cd(II) group).

hepatic damage induced by Cd(II). In contrast, Ay administration decreased the levels of LPO and PCO. Ay is a flavonoid that has been reported in view of its powerful antioxidant activity (Bagchi et al., 2004). Accordingly, Ay ameliorated oxidative damage caused by Cd(II) in mice liver.

Cd(II) 0.124 32.107 29.057 23.483 13.338

± ± ± ± ±

0.006 5.281*** 6.142 4.874# 3.175##

Zn 211.83 121.18 172.89 186.5 201.54

± ± ± ± ±

Ca 32.16 19.53** 25.32# 30.45# 26.73##

52.2 151.5 76.9 75.8 58.1

± ± ± ± ±

6.78 9.62* 7.56# 10.1# 8.34#

p < 0.05 vs. Control group. p < 0.01 vs. Control group. p < 0.001 vs. Control group. p < 0.05 vs. Cd(II) group. p < 0.01 vs. Cd(II) group.

The internucleosomal DNA cleavage with the production of oligonucleosomal fragments was the first biochemical event identified in apoptosis and it occurs almost in all instances of apoptosis Cd(II) exposure result in an increased DNA fragmentation. Conversing, co-treatment with Ay reduced the extent of DNA fragmentation, probably due to its free radical scavenging abilities because they react with carbon- and oxygen-centered radicals, including superoxide anion, thereby preventing these radicals from reaching and reacting with DNA. The induction of oxidative stress by Cd(II) is also interpreted by its impairment effects on antioxidant defense

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Fig. 8 – Metal chelation effect of different concentrations of Ay in ferrous ions.

system, such as, SOD, CAT and GSH (El-Sokkary et al., 2010). In this study, the exposure to Cd(II) was found to trigger a loss of SOD in liver. Since SOD contains Zn and Cu in its active site, the decrease of SOD activity in Cd(II)-exposed mice may be due to a Cd(II)/enzyme interaction which causes perturbations in the enzyme topography critical for its catalytic function (Casalino et al., 2002), further leading to a decreased availability of these trace elements as a result of their immobilization in the form bound to metallothionein (Brzóska et al., 2002). Similarly, reduced CAT activity has been obtained in this study which is accordance with those of Casalino et al. (2002). The

activities of SOD and CAT, which inhibited by Cd(II), are favorably restored or influenced by Ay, which is known to increase SOD and CAT activities (Chiang et al., 2006). Hence, protective effects exerted by Ay are mediated through decreases in free-radical generation as well as increases in SOD and CAT activities. In Cd(II)-induced cellular responses, GSH plays a dual role as it neutralizes ROS but also detoxifies Cd(II) directly. During the beginning of Cd(II) exposure, GSH contents undergo a sharp depletion, and then, lead to an elevation via increased GSH synthesis to diminish the oxidative damage by Cd(II). It was suggested that the enhancement of GSH content is a liverresponse to decrease the susceptibility to free radical damage and to promote the detoxification process in this organ. Our study found that exposure to Cd(II) increase GSH level, which is in consonance with previous studies (Yamano et al., 1998). The effect of Ay to restore GSH level to normal probably due to its protective action against oxidative stress. In this study, notable changes, including severe necrotic changes, inflammatory cell infiltration and vacuolization can be observed in mice liver following Cd(II) exposure at the present dose and duration. The accumulated hydroperoxides can cause cytotoxicity, which is associated with the peroxidation of membrane phospholipids by lipid hydroperoxides, the basis for hepatocellular damage. Ay administration can appreciably reduce the histological changes provoked by Cd(II) and restore to normal state, which provide direct evidence for the protective effect of Ay against Cd(II)-induced tissue injury. In addition to oxidative stress, the proposed mechanisms for Cd(II) hepatic toxicity may relate to the disturbance of essential metals (Arroyo et al., 2012). Cd(II) can compete with those biologically essential metals, like calcium, zinc and

Fig. 9 – Proposed mechanisms of Ay-exerted hepatoprotective effect. (a) Ay function as an antioxidant by activating antioxidative enzymes or quenching free radicals. (b) Ay act as a chelator by binding with Cd(II) or Fe ions, hence, reducing the formation of ROS and restore the GSH levels. (c) Ay exhibited its anti-inflammatory effect via inhibiting NO release, thereby, inactivating the NF-␬B, which involved in the inflammatory events. (d) Ay also stable the intracellular Zn or Ca levels, therefore, restore the SOD or CAT activities and inhibit the activation of NF-␬B. The combination of a–d pathways results in the decrease of oxidative parameters, and then, Ay shows its hepatoprotective effect.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

copper, to use their transport pathways. It has been well established that Cd(II) uptake through calcium transport pathways is an important biological process leading to Cd(II) hepatotoxicity. Once entering the cell, Cd(II) ions can mimic the action of other divalent ions (i.e. Zn2+ , Ca2+ ), which are employed in activating or inhibiting the action of various enzymes, leading to the interference of cell metabolism. The depletion of Zn can aggregate the Cd(II)-induced caspase-3 activation and cell apoptosis by influencing the expression of transcription factors p53 and p21 (Fernandez et al., 2003). Moreover, the release of Ca2+ to cytosol can promote the damage to mitochondria caused by Cd(II), thus mediating Cytc release and caspase-9 activation (Biagioli et al., 2008). In this study, the increase of Cd(II) ion level was associated with decreased Zn ion concentration and elevated Ca levels in liver tissue, the co-treatment of Ay could restore these changes to normal levels. NO is a highly reactive endogenous chemical produced by activated macrophages and combine with superoxide to form peroxynitrite anion to induce LPO. The intoxication of Cd(II) may alter the NO level in endothelial cell and increase the generation of RNS leading to lipid peroxidation (Chen et al., 2003; Cavicchi et al., 2000). Furthermore, NO may be involved in Cd(II) toxicity in hepatocytes by replacing Cd(II) from MT and releasing free Cd(II) to induce DNA damage, subsequently resulting in cellular growth arrest (Misra et al., 1996). In agreement with the earlier finding, our data also show that the exposure to Cd(II) has significantly increased the level of NO, however, the effect of Ay can reverse this alteration by directly reacting with NO. Based on the above results, the beneficial effect of Ay on liver is undoubtedly heterogeneous (Fig. 9): (1) in view of the consideration that oxidative stress was involved in the intoxication of liver induced by Cd(II), the antioxidant properties of Ay may play a role in mediating oxidative damage. It was well known that Ay possess the strongest antioxidant capacity, probably owing to its similar structure to flavone. (2) Ay acts as a metal-chelating molecule to modulate metal homeostasis and inhibit the occurrence of Fenton reaction by decreasing Cd(II) ion overload in liver tissue; (3) Ay increase the resistance of hepatocytes to oxidation by activating liver antioxidative enzymes and lower the GSH concentration in the liver. (4) Ay show the anti-inflammatory capacity by reducing NO content. (5) Ay restore the alteration of intracellular Zn and Ca levels to exert its hepatoprotective effect. Although Kowalczyk et al. (2003) have reported a protective action of Ay in Cd(II)-induced sub-chronic hepatotoxicity. However, their duration of treatment (30 days), way of administration (in the form of aqueous solution), and dose of Cd(II) and antioxidant (Cd(II) 4 ␮g/kg; Ay 10 mg/kg) are very different from those used in this study. We considered that all these differences (way of Cd(II) administration, dose, and duration of treatment) may attribute to a distinct effect of Ay on Cd(II)induced acute hepatotoxicity. Our study provided convincing evidences that Ay exhibit the ability to protect liver against Cd(II) damage. Possible pathways have been proposed. In combination with what reported by Kowalczyk et al. (2003), the findings in current study will provide theoretical basis for the potential of Ay extracted from blueberry as a cost-effective and safety agent in the therapy of either acute or chronic Cd(II) toxicity.

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Conflict of interest The authors declare that there are no conflicts of interest.

Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgements This work was supported in part by grants from State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (No. KF201205) and from Natural Science foundation of Shaanxi Province (No. 2012JQ2011).

references

Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Arroyo, V.S., Flores, K.M., Ortiz, L.B., Gómez-Quiroz, L.E., Gutiérrez-Ruiz, M.C., 2012. Liver and cadmium toxicity. J. Drug Metab. Toxicol., S5:001. Bagchi, D., Sen, C.K., Bagchi, M., Atalay, M., 2004. Anti-angiogenic, antioxidant, and anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula. Biochemistry (Moscow) 69, 75–80. Barnes, J.S., Nguyen, H.P., Shen, S.J., Schug, K.A., 2009. General method for extraction of blueberry anthocyanins and identification using high performance liquid chromatography–electrospray ionization-ion trap-time of flight-mass spectrometry. J. Chromatogr. A 1216, 4728–4735. Barros, D., Amaral, O.B., Izquierdo, I., Geracitano, L., Raseira, M.D.C.B., Amélia Teresinha Henriques, A.T., Ramirez, M.R., 2006. Behavioral and genoprotective effects of Vaccinium berries intake in mice. Pharmacol. Biochem. Behav. 84, 229–234. Biagioli, M., Pifferi, S., Ragghianti, M., Bucci, S., Rizzuto, R., Pinton, P., 2008. Endoplasmic reticulum stress and alteration in calcium homeostasis are involved in cadmium-induced apoptosis. Cell Calcium 43, 184–195. Borges, L.P., Brandão, R., Godoi, B., Nogueira, C.W., Zeni, G., 2008. Oral administration of diphenyl diselenide protects against cadmium-induced liver damage in rats. Chem. Biol. Interact. 171, 15–25. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Brzóska, M.M., Moniuszko-Jakoniuk, J., Jurczuk, M., Gałazyn-Sidorczuk, M., 2002. Cadmium turnover and changes of zinc and copper body status of rats continuously exposed to cadmium and ethanol. Alcohol Alcohol. 37, 213–221. Casalino, E., Calzaretti, G., Sblano, C., Landriscina, C., 2002. Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicology 179, 37–50. Cavicchi, M., Gibbs, L., Whittle, B.J., 2000. Inhibition of inducible nitric oxide synthase in the human intestinal epithelial cell line, DLD-1, by the inducers of heme oxygenase 1, bismuth salts, heme, and nitric oxide donors. Gut 47, 771–778. Chen, L., Zhou, J., Gao, W., Jiang, Y.Z., 2003. Action of NO and TNF-alpha release of rats with cadmium loading in

1026

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

malfunction of multiple system organ. Sheng Li Xue Bao 25 (55), 535–540. Chiang, A.N., Wu, H.L., Yeh, H.I., Chu, C.S., Lin, H.C., Lee, W.C., 2006. Antioxidant effects of black rice extract through the induction of superoxide dismutase and catalase activities. Lipids 41, 797–803. Choi, J.H., Choi, C.Y., Lee, K.J., Hwang, Y.P., Chung, Y.C., Jeong, H.G., 2009. Hepatoprotective effects of an anthocyanins fraction from purple-fleshed sweet potato against acetaminophen-induced liver damage in mice. J. Med. Food. 12, 320–326. Cuypers, A., Plusquin, M., Remans, T., Jozefczak, M., Keunen, E., Gielen, H., Opdenakker, K., Nair, A.R., Munters, E., Artois, T.J., Nawrot, T., Vangronsveld, J., Smeets, K., 2010. Cadmium stress: an oxidative challenge. Biometals 23, 927–940. Dinis, T.C.P., Madeira, V.M.C., Almeida, L.M., 1994. Action of phenolic derivates (acetaminophen, salicylate, and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophys. 315, 161–169. El-Sokkary, G.H., Nafady, A.A., Shabash, E.H., 2010. Melatonin administration ameliorates cadmium-induced oxidative stress and morphological changes in the liver of rat. Ecotoxicol. Environ. Safe. 73, 456–463. Fernandez, E.L., Gustafson, A.L., Andersson, M., Hellman, B., Dencker, L., 2003. Cadmium-induced changes in apoptotic gene expression levels and DNA damage in mouse embryos are blocked by zinc. Toxicol. Sci. 76, 162–170. Fowler, B.A., 2009. Monitoring of human populations for early markers of cadmium toxicity: a review. Toxicol. Appl. Pharm. 238, 294–300. Galli, R.L., Bielinski, D.F., Szprengiel, A., Shukitt-Hale, B., Joseph, J.A., 2006. Blueberry supplemented diet reverses age-related decline in hippocampal HSP70 neuroprotection. Neurobiol. Aging 27, 344–350. Gong, P., Chen, F.X., Ma, G.F., Feng, Y., Zhao, Q., Wang, R., 2008. Endomorphin 1 effectively protects cadmium chloride-induced hepatic damage in mice. Toxicology 251, 35–44. Gong, P., Chen, F.X., Liu, X.Y., Gong, X., Wang, J., Ma, Y.M., 2012. Protective effect of caffeic acid phenethyl ester against cadmium-induced renal damage in mice. J. Toxicol. Sci. 37, 415–425. Graf, B.A., Milbury, P.E., Blumberg, J.B., 2005. Flavonols, flavones, flavanones, and human health: epidemiological evidence. J. Med. Food. 8 (3), 281–290. ˘ Gülc¸in, I., Beydemir, S., Alici, H.A., Elmastas, M., Büyükokuroglu, M.E., 2004. In vitro antioxidant properties of morphine. Pharmacol. Res. 49, 59–66. Jarup, L., Åkesson, A., 2009. Current status of cadmium as an environmental health problem. Toxicol. Appl. Pharm. 238, 201–208. Jurczuk, M., Brzóska, M.M., Rogalska, J., Moniuszko-Jakoniuk, J., 2003. Iron body status of rats chronically exposed to cadmium and ethanol. Alcohol Alcohol. 38, 202–207. Karlsen, A., Retterstøl, L., Laake, P., Paur, I., Kjølsrud-Bøhn, S., Sandvik, L., Blomhoff, R., 2007. Anthocyanins inhibit nuclear factor-kappaB activation in monocytes and reduce plasma concentrations of pro-inflammatory mediators in healthy adults. J. Nutr. 137, 1951–1954. Kowalczyk, E., Kopff, A., Fijalkowski, P., Kopff, M., Niedworok, J., Błaszczyk, J., Kêdziora, J., Tyoelerowicz, P., 2003. Effect of anthocyanins on selected biochemical parameters in rats exposed to cadmium. Acta Biochim. Pol. 50, 543–548. Lau, F.C., Shukitt-Hale, B., Joseph, J.A., 2005. The beneficial effects of fruit polyphenols on brain aging. Neurobiol. Aging 26 (Suppl. 1), 128–132.

Levine, R.L., Berlett, B.S., Moskovitz, J., Mosoni, L., Stadtman, E.R., 1999. Methionine residues may protect proteins from critical oxidative damage. Mech. Ageing Dev. 107, 323–332. Lin, K.T., Xue, J.Y., Sun, F.F., Wong, P.Y.K., 1997. Reactive oxygen species participate in peroxinitrile induced apoptosis in HL 60 cells. Biochem. Biophys. Res. Commun. 230, 115–119. Lohachoompol, V., Mulholland, M., Srzednicki, G., Craske, J., 2008. Determination of anthocyanins in various cultivars of high bush and rabbit eye blueberries. Food Chem. 111, 249–254. Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474. Matés, J.M., Segura, J.A., Alonso, F.J., Márquez, J., 2010. Roles of dioxins and heavy metals in cancer and neurological diseases using ROS-mediated mechanisms. Free Radic. Biol. Med. 49, 1328–1341. Mcdougall, G.J., Ross, H.A., Ikeji, M., Stewart, D., 2008. Berry extracts exert different antiproliferative effects against cervical and colon cancer cells grown in vitro. J. Agric. Food Chem. 56 (9), 3016–3023. Misra, R.R., Hochadel, J.F., Smith, G.T., Cook, J.C., Waalkes, M.P., et al., 1996. Evidence that nitric oxide enhances cadmium toxicity by displacing the metal from metallothionein. Chem. Res. Toxicol. 9, 326–332. Moron, M.S., Despierre, J.W., Minnervik, B., 1979. Levels of glutathione, glutathione reductase and glutathione-S-transferase activities in rat lung and liver. Biochim. Biophys. Acta 582, 67–78. Nemmiche, S., Chabane-Sari, D., Guiraud, P., 2007. Role of alpha-tocopherol in cadmium-induced oxidative stress in Wistar rat’s blood, liver and brain. Chem. Biol. Interact. 170, 221–230. Neto, C.C., 2007. Cranberry and blueberry: evidence for protective effects against cancer and vascular diseases. Mol. Nutr. Food Res. 51, 652–664. Newairy, A.A., El-Sharaky, A.S., Badreldeen, M.M., Eweda, S.M., Sheweita, S.A., 2007. The hepatoprotective effects of selenium against cadmium toxicity in rats. Toxicology 242, 23–30. Niehius, W.G., Samuelson, B., 1968. Formation of malondialdehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur. J. Biochem. 6, 126–130. Reen, R.K., Nines, R., Stoner, G.D., 2006. Modulation of N-nitrosomethylbenzylamine metabolism by black raspberries in the esophagus and liver of Fischer 344 rats. Nutr. Cancer 54 (1), 47–57. Reitman, S., Frankel, S., 1957. A colorimetric method for determination of serum GOT and GPT. Am. J. Clin. Pathol. 28, 56–63. Rimando, A.M., Kalt, W., Magee, J.B., Dewey, J., Ballington, J.R., 2004. Resveratrol, pterostilbene, and piceatannol in vaccinium berries. J. Agric. Food Chem. 52, 4713–4719. Santos, F.W., Zeni, G., Rocha, J.B.T., Weis, S.N., Fachinetto, J.M., Favero, A.M., Nogueira, C.W., 2005. Diphenyl diselenide reverses cadmium-induced oxidative damage on mice tissues. Chem. Biol. Interact. 151, 159–165. Schmidt, B.M., Erdman Jr., J.W., Lila, M.A., 2006. Differential effects of blueberry proanthocyanidins on androgen sensitive and insensitive human prostate cancer cell lines. Cancer Lett. 231 (2), 240–246. Sellins, K.S., Cohen, J.J., 1987. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J. Immunol. 139, 3199–3206. Shih, P.H., Yeh, C.T., Yen, G.C., 2005. Effects of anthocyanidin on the inhibition of proliferation and induction of apoptosis in human gastric adenocarcinoma cells. Food Chem. Toxicol. 43, 1557–1566.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 1015–1027

Shih, P.H., Yeh, C.T., Yen, G.C., 2007. Anthocyanins induce the activation of phase II enzymes through the antioxidant response element pathway against oxidative stress-induced apoptosis. J. Agric. Food Chem. 55, 9427–9435. Stohs, S.J., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336. Tsuda, T., Horio, F., Uchida, K., Aoki, H., Osawa, T., 2003. Dietary cyanidin-O-beta-d-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. J. Nutr. 133, 2125–2130. Tzirogiannis, K.N., Panoutsopoulos, G.I., Hereti, R.I., Alexandropoulou, K.N., Basayannis, A.C., Mykoniatis, M.G.,

1027

2003. Time-course of cadmium-induced acute hepatotoxicity in the rat liver: the role of apoptosis. Arch. Toxicol. 77, 694–701. Yamano, T., Shimizu, M., Noda, T., 1998. Age-related change in cadmium-induced hepatotoxicity in Wistar rats: role of Kupffer cells and neutrophils. Toxicol. Appl. Pharmacol. 151, 9–15. Yao, N., Lan, F., He, R.R., Kurihara, H., 2010. Protective effects of bilberry (Vaccinium myrtillus L.) extract against endotoxin-induced uveitis in mice. J. Agric. Food Chem. 58 (8), 4731–4736.