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
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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).
1020
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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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
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 #
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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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 #
