Journal of Hazardous Materials 274 (2014) 247–257

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Protective role of oligomeric proanthocyanidin complex against hazardous nodularin-induced oxidative toxicity in Carassius auratus lymphocytes Hangjun Zhang ∗ , Wendi Fang, Wenfeng Xiao, Liping Lu, Xiuying Jia Department of Environmental Sciences, Hangzhou Normal University, Xuelin Road 16#, Xiasha Gaojiao Dongqu, Hangzhou 310036, Zhejiang Province, China

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• OPC can inhibit NOD-induced toxicity of fish lymphocytes by lowering the risk of oxidative stress. • OPC can reduce NOD-induced toxicity by mediating the expression of bcl-2 family proteins. • Physical toxicity of NOD is reduced after conjugating with GSH. • OPC can be acted as a protective agent to alleviate NOD-induced toxicity in C. auratus lymphocytes.

a r t i c l e

i n f o

Article history: Received 18 December 2013 Received in revised form 9 April 2014 Accepted 14 April 2014 Available online 21 April 2014 Keywords: Proanthocyanidin Nodularin Lymphocytes Antiapoptosis Oxidative stress

a b s t r a c t Nodularin (NOD) is a hazardous material widely detected in water blooms. Fish immune cells are extremely vulnerable to NOD-induced oxidative stress. Oligomeric proanthocyanidin complex (OPC), extracted from grapeseed, was used as an antioxidant to eliminate reactive oxygen species and prevent apoptotic effects. Carassius auratus lymphocytes were treated with different concentrations (0, 10, 100, and 1000 ␮g/L) of OPC and a constant dose (100 ␮g/L) of NOD for 12 h in vitro. OPC inhibited mitosis by decreasing intracellular levels of oxidative stress, regulating antioxidant enzymes (CAT, SOD, GPx, GR, and GST), mediating bcl-2 family proteins, and deactivating caspase-3. Glutathione (GSH) levels in group V (NOD 100 ␮g/L; OPC 1000 ␮g/L) showed a twofold increase compared with corresponding levels in group II (NOD 100 ␮g/L). Structure parameters of NOD and NOD-GSH were calculated using SYBYL 7.1 software. C log P and HINK log P values of NOD-GSH decreased by 10.4- and 2.3-fold, respectively, compared with corresponding values of NOD. OPC-stimulated GSH can lower the lipophilicity and polarity of NOD. OPC, as a protective agent, can alleviate NOD-induced toxicity in C. auratus lymphocytes by regulating oxidative stress and inducing NOD-GSH detoxification. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Several bloom-forming species of cyanobacteria can produce hazardous cyanotoxins as secondary metabolites [1]. Among these

∗ Corresponding author. Tel.: +86 571 28867058; fax: +86 571 28865327. E-mail addresses: [email protected], [email protected] (H. Zhang). http://dx.doi.org/10.1016/j.jhazmat.2014.04.020 0304-3894/© 2014 Elsevier B.V. All rights reserved.

cyanotoxins, microcystin (MC) and nodularin (NOD) are the most essential cyanotoxins currently under study. MC and NOD are considered ubiquitous contaminants of surface water worldwide, elevating risks for aquatic organisms and humans at low doses [2,3]. Therefore, reducing the hazards of MC and NOD to wild and domestic animals, as well as humans, is of primary importance. NOD is a cyclic pentapeptide with a structure similar to that of MC. This structure consists of Adda, d-glutamic acid, N-methyldehydrobutyrine, d-erythro-␤-methylaspartic acid, and

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Fig. 1. The chemical structure of NOD.

l-arginine [4–6] (Fig. 1). Adda in NOD damages the liver by inhibiting protein phosphatase (PP) catalytic subunit types 1 and 2A via a mechanism identical to that of MC [7–9]. Fish immune cells are also extremely vulnerable to NOD. NOD may weaken the immune system of dogs [10] and can induce the mitochondrial apoptotic pathway [11]. MC also induces the intracellular production of reactive oxygen species (ROS) [12], thereby enhancing oxidative stress markers in fish [13] and mammals [14]. Based on similarities between the structures and toxicities of MC and NOD [4–9], we hypothesized that antioxidants exert the same effects on NOD and MC, especially in the immune system. Reductions in immune responses may be associated with the longstanding and acute exposure of lymphocytes to oxidative stress, leading to the loss of transcription factor activity and diminished cytokine production in response to antibody generator stimulation [15–17]. Cells that engulf the foreign invader can produce ROS and other chemicals in the phagosome to kill such invaders. Sufficiently high levels of oxidative stress harm certain host tissues and induce the lymphocyte apoptosis. As such, research efforts have focused on reducing MC toxicity by decreasing intracellular oxidative stress through the antioxidant defense system and modulating glutathione (GSH) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), or glutathione S-transferase (GST) activities [18]. Certain antioxidants reduce MC-induced damage even when the latter is administered at toxic doses. Vitamin E [19–21], vitamin C [20,21], lycopene [22], tea polyphenols [23], flavonoids [24], and melatonin [20] in mice; vitamin E [25] in fish; and vitamin E [26] and lipoic acid [27] in carp have been proven to present chemoprotective roles against MC-induced toxicity. The MC-induced apoptotic signaling pathway has been summarized and induction of apoptosis in vitro in MC-exposed hepatocytes has been demonstrated to be closely linked to ROS generation [28,29]. Ding et al. [12] proposed that MC can cause cell death through at least three pathways: altering GSH depletion, disrupting mitochondrial electron transport chain, and inducing cellular protein phosphorylation; these pathways followed by intracellular oxidative stress and oxidative damage. Percival [30] reported that bioactive compounds in grape can support or maintain the immunity responses of animals and humans, particularly by repairing oxidative damage. In our research, oligomeric proanthocyanidin complex (OPC), a compounds extracted from grape seeds, was used as an antioxidant to reduce NOD toxicity by stimulating GSH production and maintaining the mitochondrial

membrane potential (MMP). We also calculated the quantum chemical properties of MC and NOD and their conjugation with GSH. GSH can reduce NOD-induced oxidative stress effects and change the structures of MC and NOD by the conjugation [29,12]. Such changes in structure may result in reducing NOD toxicity [31]. Our previous study has demonstrated that the mitochondriamediated pathway is key to the regulation of cytotoxicity in fish lymphocytes [11]. In the present study, the protective roles of OPC against NOD-induced oxidative stress and apoptosis are achieved by reduction of apoptotic cells, the enhancement of MMP, inhibition of the fluctuations in ROS levels and malondialdehyde (MDA) contents, mediation of CAT, SOD, GST, glutathione reductase (GR), and GPx activities, enhancement of GSH, and regulation of bcl-2, bax and caspase-3 protein expression. Quantum chemical parameters calculation demonstrate that GSH can obviously reduce NOD toxicity. 2. Materials and methods 2.1. Chemicals and toxin Purified NOD (CAS No. 118399-22-7, C41 H60 N8 O10 , MW = 824.96) and OPC (CAS No. 131631-89-5, C26 H31 N3 O4 , MW = 449.54) were purchased from Sigma (St. Louis, MO, USA). Lymphoprep with a density of 1.077 g/mL was obtained from Huadong Pharmaceutical (Hangzhou, China). RPMI-1640 medium was obtained from Hangzhou Key Shengwu (Hangzhou, China). MDA, SOD, CAT, rhodamine 123 (Rh 123), and Fluo3-AM detection kits as well as caspase activity kits were obtained from Beyotime Institute of Biotechnology (Haimen, China). Trizol reagent and the M-MLV reverse transcriptase kit used for detecting gene expression were purchased from Invitrogen (Carlsbad, CA, USA) and Toyobo (Tokyo, Japan), respectively. bcl-2, bax, and caspase-3 antibodies as well as the horseradish peroxidase-conjugated goat anti-rabbit IgG used for Western blot analysis were obtained from Zymed (San Francisco, CA, USA) and Wuhan BoshiDeBiotech (Wuhan, China). All other chemicals were obtained from Sigma, unless otherwise specified. 2.2. Experimental fish Carassius auratus (6–12 mo old) of both sexes and with average lengths of approximately 25–30 cm were obtained from the

H. Zhang et al. / Journal of Hazardous Materials 274 (2014) 247–257 Table 1 Treatment protocols of Carassius auratus lymphocytes in different groups. Treatment

Group I

II

III

NOD (␮g/L) OPC (␮g/L)

0 0

100 0

100 10

IV

V

100 100

100 1000

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4 ◦ C for 15 min to obtain the supernate. Using the assay kit, the GSH activities of the lymphocytes were determined by a fluorescence spectrophotometer (RF-3501) at 405 nm. GSH levels were quantified as described by Fernández-Checa and Kaplowitz [37]. Activity was expressed in ␮M/g protein in the samples. 2.8. Measurement of antioxidant enzymes activities

hatchery of the Freshwater Fisheries Institute of Zhejiang, China. All experimental fish were fed with pellet food at a daily ration of 0.7% of their body weight in re-circulating water controlled to 25 ± 1 ◦ C. After 10 d of rearing, healthy fish were held in the laboratory for study. 2.3. Isolation of lymphocytes and cell culture Based on the method described by Kemenade et al. [32], head kidneys containing lymphocytes were obtained from C. auratus, mixed together, washed twice in ice-cold RPMI-1640 culture medium, collected, and layered on 1.5 vol. lymphoprep. Following 30 min of centrifugation at 650 × g, the lymphocyte layer was collected and washed thrice using phosphate buffered saline (PBS). Finally, the cells were cultured in antibiotic-free RPMI-1640 medium with 5% fetal calf serum in a CO2 atmosphere of 27 ◦ C for 5 h to remove adherent cells. The collected cells are divided into five groups, and each group was treated with different concentrations of NOD and OPC (Table 1). 2.4. Apoptosis detection and propidium iodide (PI) staining for flow cytometry Lymphocytes were collected after 12 h of incubation, washed with ice-cold PBS, and fixed in 70% ethanol for at least 24 h at 4 ◦ C. Cells were washed twice with PBS and treated with 100 mg/mL RNase (Sigma) and 50 ␮g/mL PI (Sigma) staining buffer for 30 min at room temperature [33]. Cells were then filtered using a BD Falcon circular tube (No. 352235, Becton Dickinson, New Jersey, USA) prior to analysis using a Guava easyCyte 8HT flow cytometer (Merck Millipore, Darmstadt, Germany). PI was excited at 488 nm and detected at 630 nm. 2.5. ROS assay Lymphocytes exposed to NOD and OPC for 12 h were collected and homogenized in cold PBS. After centrifugation at 650 × g for 5 min, the collected lymphocytes were exposed to 10 ␮M 2 ,7 dieMorofluoreseeindiacetate at 27 ◦ C. Cells were analyzed using a Shimadzu RF-3501 fluorescence spectrophotometer (Shimadzu, Korneuburg, Austria) at an excitation wavelength of 488 nm and emission wavelength of 525 nm, as previously described [34]. 2.6. Measurement of MDA content Lymphocytes obtained from fish were collected and homogenized in cold PBS. Homogenates were centrifuged for 15 min at 650 × g and 4 ◦ C to obtain the supernate. The concentration of MDA was determined according to Ohkawa et al. [35]. MDA was analyzed using an assay kit and measured at 532 nm using a fluorescence spectrophotometer (RF-3501) according to the manufacturer’s instructions. Sample activity was expressed in nmol/mg protein.

SOD activity was measured following procedures by Mccord and Fridovich [36]. Lymphocytes obtained from fish were collected and homogenized in cold PBS. The homogenates were centrifuged at 650 × g and 4 ◦ C for 15 min to obtain the supernate. The supernate was treated with the reagents in the SOD assay kit. The treatment solution was incubated at 37 ◦ C for 10 min. Finally, SOD content was determined at 450 nm using a fluorescence spectrophotometer (RF3501) according to the manufacturer’s instructions. Sample activity was expressed in unit/mg protein. CAT, GST, GR and GPx activities were measured according to the manufacturer’s instructions of the related assay kit. 2.9. MMP determination Cytofluorometric determination of MMP was conducted according to previously described methods [38]. Treated lymphocytes were harvested, resuspended in 1 mL of old PBS, and stained with Rh123 at a final concentration of 5 ␮g/mL for 30 min at room temperature. The lymphocytes were washed with cold PBS and collected for analysis using a fluorescence spectrophotometer (RF3501) at an excitation wavelength of 507 nm and an emission wavelength of 529 nm. 2.10. Western blot analysis Lymphocyte samples (1 mL) were mixed with 9 mL of 2× SDS loading buffer (0.1 M Tris–HCl buffer at pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue, and 0.2 M dithiothreitol) and boiled for 5 min. Samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Millipore Co., Billerica, MA, USA). Blots were blocked with 1% casein in Tris buffered saline and Tween 20 (TTBS) for 3 h at 37 ◦ C and incubated with bcl-2, bax, and caspase-3 antibodies in blocking buffer at 4 ◦ C overnight. Membranes were washed thrice with TTBS before incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG for 30 min. The same process was carried out with ABC reagents in TTBS. The membrane was washed 2× 5 min in TTBS and 1× 5 min in Tris buffered saline and incubated with amino-ethyl carbazole reagent for 10 min. Finally, the membrane was washed in two changes of H2 O2 for 10 min each [39]. ␤-Actin expression was examined as the internal control. 2.11. Intracellular Ca2+ detection Lymphocytes obtained from fish were collected in cold PBS and stained with a final concentration of 5 ␮g/mL set in Fluo3-AM for 40 min at 27 ◦ C. Cells were then washed twice with cold PBS, collected for analysis on a Guava easyCyte 8HT flow cytometer in Incyte mode, and examined using previously described methods [40]. 2.12. Caspase-3 activity assay

2.7. Measurement of GSH content Lymphocytes obtained from fish were collected and homogenized in cold PBS, The homogenates were centrifuged at 650 × g at

Caspases-3 activity was determined using the caspase activity kits. Lysis buffer was added to lymphocytes after their treatment with various concentrations of OPC at the same dose of NOD. Assays

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Fig. 2. Effects of different concentrations (0, 10, 100, and 1000 ␮g/L) of OPC on the apoptotic percentage of C. auratus lymphocytes exposed to 100 ␮g/L nodularin (NOD) for 12 h. (A) Flow cytometry histograms (groups I, II, and V) indicate the percentage of cells detected in specific cell cycle phases; (B) histogram showing the percentage of apoptotic cells in all groups. Results represent the mean ± SD of five samples. (a) Group I. (b) Group II. *p < 0.05 and **p < 0.01.

were performed as previously described. Lysates were then incubated at 37 ◦ C for 2 h, and samples were detected by a microplate reader at an absorbance wavelength of 405 nm. The detailed analytical procedure is described in the manufacturer’s protocol.

presented as the mean ± SD of four independent experiments performed in duplicate and triplicate. Samples were analyzed using one-way ANOVA with statistical significance defined as p < 0.05 and p < 0.01.

2.13. Quantum chemical properties calculation

3. Results

Molecule parameters were determined using SYBYL7.1 software, and lipophilicity parameters including molecular hydrophobicity log P (P: partition coefficient in octanol/water solvent system) were obtained. C log P was empirically estimated according to Leo and Hansch [41], while HINK log P was determined using hydropathic interactions analysis [42]. Polarity parameters including charged partial surface area descriptors (CPSA) [43] and MOLPROP descriptors were determined. CPSA calculations included total surface area (total area) and total molecular volume (mol volume), among others. SYBYL7.1 MOLPROP descriptors were also calculated with a probe radius of 0 A˚ (corresponding to a simple van der Waals shell) or 1.4 A˚ (approximates the radius of water) and area resolution of 36, corresponding to a 58◦ grid. These descriptors contain Polar Surface Area (PSA) (consideration of all O, N, and S atoms as well as H covalently bonded to these atoms), surface area (AREA), polar volume (PV) (considering all O, N, and S atoms as well as H covalently bonded to these atoms), and volume (VOLU). Variations in CPSA calculations compared with MOLPROP AREA results are attributed to differences in van der Waals surface computations.

3.1. OPC effects on NOD-induced apoptosis

2.14. Statistical analysis Means and standard deviations of experimental parameters were calculated using Microsoft Excel® software. Data were

Fig. 2 shows the percentages of apoptotic cells in group treated by different concentrations (0, 10, 100, and 1000 ␮g/L) of OPC and 100 ␮g/L of NOD for 12 h in vitro. Compared with group I, the percentage of apoptotic cells of group II increased rapidly. The apoptotic percentage of lymphocytes treated with 0, 10, 100, and 1000 ␮g/L of OPC were 44.3%, 31.2%, 21.7% and 10.1%, respectively, after 12 h. The percentages of apoptotic cells decreased with increasing dosage of OPC, and the apoptotic percentage was only half of that in group VI compared with that in group II. 3.2. Effects of OPC on biochemical indicators in the cytoplasm ROS expression is an important biomarker that reflects oxidative stress levels [44]; it is considered a critical mediator of apoptosis. Fig. 3 shows that ROS levels increased 1.4-fold compared with that in group I after treatment with 100 ␮g/L NOD but decreased by addition of 1000 ␮g/L OPC. MDA, another biochemical indicator of lipid peroxides, represents an oxidation product of polyunsaturated fatty acids [45]. Fig. 4 shows that 100 ␮g/L NOD increased cellular MDA contents by 1.4-fold compared with that in group I; application of OPC did not induce a significant decrease in MDA content. GSH content showed a significant decrease after exposure to 100 ␮g/L NOD compared with group I (Table 2). However, the

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Table 2 Antioxidant enzymes activities and GSH content exposed to OPC and NOD for 12 h. Group

NOD (␮g/L)

OPC (␮g/L)

I II III IV V

– 100 100 100 100

– – 10 100 1000

GSH (␮M/g prot) 8.28 5.52 5.80 8.71 10.87

± ± ± ± ±

1.88 2.04a * 0.16 3.11 2.19a * ,b **

SOD (U/mg prot) 7.33 3.64 3.60 7.49 8.66

± ± ± ± ±

0.13 0.57a ** 0.31a ** 1.36b ** 0.51a * ,b **

CAT (U/mg prot) 9.88 5.52 5.56 10.82 13.03

± ± ± ± ±

0.85 2.03a * 1.28a ** 1.62b * 0.71a * ,b *

GST (U/mg prot) 31.67 11.80 29.02 35.47 51.71

± ± ± ± ±

3.43 3.59a ** 6.70b ** 2.23b ** 1.24a ** ,b **

GR (U/mg prot) 0.00971 0.0229 0.0132 0.0112 0.0108

± ± ± ± ±

0.00186 0.059a ** 0.0067 0.00458b * 0.00188b *

GPx (U/mg prot) 33.80 27.61 30.63 35.28 46.77

± ± ± ± ±

1.50 2.17a * 17.10 5.53 3.41a ** ,b **

Results are the mean ± SD of five samples. (a) Group I. (b) Group II. * p < 0.05. ** p < 0.01

Fig. 3. Effects of different concentrations (0, 10, 100, and 1000 ␮g/L) of OPC on reactive oxygen species (ROS) in C. auratus lymphocytes exposed to 100 ␮g/L NOD for 12 h. Results are the mean ± SD of five samples. (a) Group I. (b) Group II. *p < 0.05 and **p < 0.01.

GSH concentrations were significantly increased after addition of 1000 ␮g/L OPC. The activities of antioxidant enzymes, including SOD, CAT, GST, GR and GPx were detected. Table 2 shows that the SOD level of the treatment group incubated with 100 ␮g/L NOD decreased

to half of that in group I. A dose-dependent increase in SOD activity was observed upon addition of up to 100 ␮g/L OPC. SOD activity showed a twofold increase upon addition of OPC compared with that in group II. Moreover, SOD activities increased compared with that in group I by addition of 1000 ␮g/L OPC. In general, however, only small differences were observed between groups. GST is abundant in the cytosol and can discharge NOD from bile or urine through catalysis of thiols (–SH) of GSH to hydrophobic compounds and convert them into hydrophilic materials [46]. Table 2 shows that the activity of GST in group II decreased 62.8% compared with that in group I, but the activities of GST in group III, IV and V increased after the protective agent OPC was added. GST activity in group V significantly jumped 1.6-fold and 4.4-fold compared to that in group I and group II, respectively. In Addition, GR plays an important role in reducing glutathione disulfide (GSSG) to its sulfhydryl form, GSH; GPx, by contrast, causes the reverse reaction [47]. Table 2 shows that changes in GPx activities were similar to those of GST, where its activity in group II increased to 81.7% compared with group I, and increased by 1.4-fold for group V. By contrast, GR activities decreased after addition of OPC. In particular, the GR activity of group V decreased to half of that of group II. 3.3. Effects of OPC on proapoptotic proteins Bcl-2 proteins play an important role in mediating cell apoptosis; the expressions of these proteins were detected by Western blot. Fig. 5A shows that the relative level of bcl-2 expression in test lymphocytes significantly decreased compared with that in group I after treatment with 100 ␮g/L NOD for 12 h. After addition of OPC, bcl-2 protein expression increased sharply. At 1000 ␮g/L OPC, bcl-2 expression increased 4.8-fold compared with group II. By contrast, bax expression decreased after addition of OPC (Fig. 5B). Treatment with 1000 ␮g/L OPC decreased bax expression to half of that in the NOD-treated group. Aside from bcl-2 proteins, caspase-3 is another essential proapoptotic protein detected in the present study. Caspase has been shown to have a pivotal function in execution apoptosis induced by diverse stimuli [48]. Western blot was used to measure the expression of caspase-3 in lymphocytes after treatment with 100 ␮g/L NOD and exposure to different concentrations of OPC for 12 h. Fig. 5C shows a significant increase in caspase3 expression before addition of OPC and a significant decrease after addition of OPC. After addition of 1000 ␮g/L OPC, caspase3 expression decreased to 37% compared with that in group II. 3.4. Determination of mitochondrial membrane potential (MMP) and intracellular Ca2+

Fig. 4. Effects of different concentrations (0, 10, 100, and 1000 ␮g/L) of OPC on MDA content in C. auratus lymphocytes exposed to 100 ␮g/L nodularin (NOD) for 12 h. Results are the mean ± SD of five samples. (a) Group I. (b) Group II. *p < 0.05 and **p < 0.01.

Loss of MMP is one of several key events that occur in the mitochondria during early stages of apoptosis [49]. Reductions in MMP, as evaluated by the MMP-dependent fluorochrome Rh123,

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Fig. 5. Expressions of bcl-2 (A), bax (B), and caspase-3 (C) in C. auratus lymphocytes exposed to 100 ␮g/L NOD and different concentrations (0, 10, 100, and 1000 ␮g/L) of OPC for 12 h; ␤-actin was used as the internal control. The representative autoradiograph (A–C) and the intensities of protein bands (D) were quantified by densitometry. Results are the mean ± SD of five samples. (a) Group I. (b) Group II. *p < 0.05 and **p < 0.01.

have been shown to indicate apoptotic prophase. Compared with that of group I, the MMP of group II decreased by 20% after 12 h. OPC helped cells exposed to 100 ␮g/L NOD rapidly recover from lost MMP (Fig. 6).

Intracellular Ca2+ is considered a mediator of signal transduction leading to apoptosis [50]. Cytosolic Ca2+ levels were initially detected after exposure to 100 ␮g/L of NOD. Fig. 7 illustrates a significant increase in intracellular Ca2+ in lymphocytes exposed

Fig. 6. NOD-induced cytotoxicity in C. auratus lymphocytes estimated by MMP assay. Lymphocytes were incubated in the presence of OPC at 0, 10, 100, and 1000 ␮g/L for 12 h with 100 ␮g/L of NOD. Results are the mean ± SD of five samples. (a) Group I. (b) Group II. *p < 0.05 and **p < 0.01.

Fig. 7. Effects of different concentrations (0, 10, 100, and 1000 ␮g/L) of OPC on Ca2+ content in C. auratus lymphocytes exposed to 100 ␮g/L NOD for 12 h. Results are the mean ± SD of five samples. (a) Group I. (b) Group II. *p < 0.05 and **p < 0.01.

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Table 3 Physical properties of MCLR, MCLR-GSH, NOD, NOD-GSH. Chemicals

MCLR MCLR-GSH NOD NOD-GSH

Calculated physical properties C log P

HINK log P

Total area

Mol volume

PSAa

AREAb

RPSV

PVc

VOLUd

RPV

0.68 −2.72 −0.42 −4.35

−3.37 −6.71 −3.07 −6.94

1164 1512 1169 1591

2461 3104 2189 2869

288.159 518.717 351.081 645.792

1164.727 1492.538 1162.184 1593.667

24.74% 34.75% 30.21% 40.52%

508.493 701.051 505.169 804.269

2461.108 3103.681 2189.068 2869.048

20.66% 22.59% 23.08% 28.03%

RPSV: the ratio of polar surface area on molecular surface area; RPV: the ratio of polar volume on volume. a Molprop polar surface area, probe radius = 1.4 A˚ approximating water radius. b ˚ Molprop molecular surface area, probe radius = 1.4 A. c ˚ Molprop polar volume, probe radius = 1.4 A. d ˚ Molprop volume, probe radius = 1.4 A.

to 100 ␮g/L NOD. Increasing OPC concentrations did not decrease cytosolic Ca2+ levels significantly. 3.5. Quantum chemical properties The structural parameters of microcystin-LR (MC-LR), MCLRGSH, NOD, and NOD-GSH were calculated and results are displayed in Table 3. C log P and HINK log P, which are expressed as partition coefficients in octanol/water, reflect the lipophilicity of a compound, while polarity may be described by PSA, RPSA, PV, and RPV; relevant results are shown in Table 3. Table 3 shows that C log P and HINK log P decreased after MC-LR or NOD binding with GSH, which indicates that GSH can lower the lipophilicity and polarity of these toxins. 4. Discussion NOD, which was first detected from Nodularia spumigena blooms, can accumulate in tissues of various fish species [51,52]. NOD mediates oxidative stress in cells by reducing the activities of several antioxidant enzymes, such as CAT, GST in the cytosol [53], and inducing the mitochondrial apoptosis pathway. The immune system, in particular, is a susceptive target of higher levels of ROS, and vulnerable to NOD attack [11]. In the present study, C. auratus lymphocytes were used as in vitro test model and NOD treatment in group II showed a 16.6-fold increase in apoptotic percentage (Fig. 2), 1.4-fold increases in ROS levels and MDA content (Figs. 3 and 4), 50% decreases in SOD and CAT activities (Table 2), and 33% decreases in GSH content (Table 2) compared with group I. These results are consistent with earlier research showing that NOD triggers cell apoptosis in mice, fish, and mussels with increasing levels of oxidative stress [51,11,54,55]. Thus, we chose antioxidant OPC as a protective agent to defend lymphocytes against NOD-induced apoptosis. Bioactive compounds (flavonoids, naringin) in grapes can protect hepatocytes against MC-induced cell death [56,57] and defend the immunity responses of animals and humans against oxidative damage [28]. Dogan and Celik [58] used GS to protect rat hepatocytes from ethanol-induced oxidative stress and found reductions in MDA content and induced SOD activity after treatment with GS, similar to Ulusoy’s data [59] on MDA content. As shown in Figs. 3 and 4, ROS levels and MDA contents decreased significantly compared with group II after treatment with up to 100 ␮g/L OPC (Fig. 4). Table 2 shows a notable increase in SOD, CAT, GST, and GPx activities after addition of 1000 ␮g/L OPC to lymphocytes; oxidative stress brought about by NOD-induced increases in ROS also decreased. Compared with other antioxidants, OPC greatly affected several biochemical indicators, thereby confirming its potential use as a protective agent for resisting cyanotoxin-induced oxidative stress (Table 4). Besides efficiently affecting antioxidant enzymes activities, OPC also significantly elevated GSH levels. From Table 2, GSH level in group V increased 1.9-fold compared with that in

group II, even 1.3-fold compared with that in group I. These results demonstrate that GSH content is sensitive to the OPC dosage and that OPC inhibits NOD-induced apoptosis by releasing an abundance of GSH. Although GSH has been shown to present an important function in OPC rescue of lymphocytes from NOD-induced oxidative stress, GSH levels do not always increase with increasing OPC concentration. Thus, the OPC concentration is a critical consideration and must be optimized. Wang et al. [60] reported that higher concentrations of OPC (higher than 1000 ␮g/L) induce lymphocyte apoptosis by depleting intracellular thiols. Table 2 also shows that GR activities decrease to group I levels after addition of OPC. Table 2 reveals that OPC indirectly controls GPx activity by increasing GSH levels, which results in decreases in thiol concentration [60]. Results of a study by Wang et al. [60] and the present study indicate that OPC stimulates –SH to form GSH, which then converts to GSSG via GPx. However, GPx activation does not guarantee that GR can disrupt the GSH/GSSH balance, which results in apoptosis. Therefore, an appropriate concentration of OPC is key to ensure adequate GSH contents to alleviate NOD-induced toxicity. In this research, we found that 1000 ␮g/L OPC effectively reduces NOD-induced oxidative stress. Mitochondria, which are major generators of ROS, are sensitive to damage by ROS, especially at high levels. Fig. 5 shows that the MMP of cells treated with NOD increased whereas the concentration of ROS decreased after addition of 1000 ␮g/L OPC. Furthermore, OPC modulated MMP levels by mediating biochemical indicators in the cytoplasm as well as proapoptotic members of the bcl-2 proteins. bcl-2 proteins regulate the mitochondrial pathway of apoptosis by joint actions directly on the mitochondrial membrane or indirectly on cytosolic Ca2+ . In the direct method, conformational changes in bax allow the molecule to insert into the mitochondrial membrane as a homooligomerized multimer, which results in the formation of large protein-permeable pores and decreases MMP levels [61]. bcl-2 residing in the outer mitochondrial membrane can protect mitochondria against MMP reduction, presumably by binding to and neutralizing other proapoptotic proteins [62]. Figs. 6 and 7 clearly show that increased dosages of OPC induce bcl-2 expression and reduce bax expression, respectively. This effect is most significant when the concentration of OPC reaches 1000 ␮g/L. Pinton and et al. [63] found that Ca2+ movement from the endoplasmic reticulum to the mitochondria is a key process that leads to apoptosis. Several authors have also demonstrated that embryonic fibroblasts from mice featuring deleted proapoptotic members of the bcl-2 family, namely, bax and bak, display reduced [Ca2+ ]ER [64]. Fig. 7 shows that in all OPC treatment groups decreases in cytosolic Ca2+ levels are insignificant compared with those in group I. Previous studies have found that loss of MMP leads to the release of proapoptotic factors, such as cytochrome c, which can activate apoptosis-protease activating factor 1. This action generates the complex known as caspase-3, which promotes the simultaneous release of cytochrome c [65]. Fig. 5 (C) shows that 1000 ␮g/L OPC

254

Table 4 Summary of the effects of different antioxidants on the biomarkers of cyanotoxin-induced detoxification and oxidative stress in vertebrates. Exposure duration and dosage of antioxidant

Lycopene

Pre-treated for 2 weeks; 10 mg/mouse/d

Green tea polyphenols

Intragastric administration for 0–18 days; concentrations: 50, 100, 200 mg/kg/day

Quercetin

Pre-treated for 1, 3 days; 200 mg/kg

Cyanotoxins

MC-LR Vitamin C and E

Vitamin E

Oral administration for 3 days; 250 mg/kg/day (vitamin C) and 200 mg/kg/day (vitamin E) Fed for 4 weeks; 8.33, 33.3 U/mouse/day

Fed for 0–7 days; dose of 200,700 mg/kg diet

Exposure duration and dosage of cyanotoxins

Targets

Parameter

Observed change of parameters

Reference

Chronic—injection for 2 weeks; single dose 75 ␮g/kg Acute—injection for 6–18 days; single dose 10 ␮g/kg/day

Mice: liver

TBA



[22]

Mice: liver, serum

SOD, GSH, bcl-2 MDA, APP(liver) CAT

 

[23]



[24]

MMP ROS, MDA, bax TBA

  

    (at 200 mg/kg diet)  

[25]

Fish: gill

LPO, CAT SOD LPO SOD CAT LPO, SOD, CAT

Mice: liver

SOD, CAT, GPx



[77]

Fish: lymphocytes

SOD, CAT, MMP, bcl-2, ROS, MDA, bax, caspase-3, APP, Ca2+



Acute—exposed for 1, 3 days; 57.5 g/kg Acute—treatment for 12 h; single dose 60 g/kg Chronic—injection for every 3 days from day 8; single dose 53.7 g/kg Acute—exposed for 24 h; single dose 120.0 ␮g

Mice: serum Mice: liver Mice: liver

Fish: liver Fish: kidney

Melatonin

Fed for 7 days; dose of 5, 10, 15 mg/kg/d NOD

OPC

Treated for 12 h; 10, 100, 1000 ␮g/L

Acute—fed for 7 days; single dose 5 lg/kg/d Acute—exposed for 12 h; single dose 100 ␮g/L

[25] [19]

This study



Symbols and abbreviations: , inhibition in comparison with control; , stimulation; , no effect; TAB, thiobarbituric acid; GSH, glutathione; GPx, glutathione peroxidase; LOP, lipid peroxidation; APP, apoptosis percentage.

H. Zhang et al. / Journal of Hazardous Materials 274 (2014) 247–257

Antioxidant

H. Zhang et al. / Journal of Hazardous Materials 274 (2014) 247–257

is an efficient dosage for alleviating NOD-induced apoptosis of lymphocytes by decreasing caspase-3 expression, which has been observed in recent research [66]. These studies indicate that OPC has antiapoptotic ability. Besides inhibiting NOD-induced oxidative stress and apoptosis, OPC may interfere with NOD uptake into lymphocytes in two ways. One possible mechanism is that OPC reacts with NOD in the cell exterior. The other is OPC inhibits the uptake mechanism present in lymphocytes. Jokela et al. [67] and Herfindal et al. [68] found that a novel cyanobacterial cyclic peptide does not interfere with the binding of cyanotoxins to PP2A, which is attributed to blockage of the hepatocyte uptake of MC and NOD by inhibiting organic anion transporter protein expression. Table 2 shows that OPC can stimulate increases in GSH. The calculated data in Table 3 further prove the conjugation between GSH and NOD. Thus, OPC may interfere with NOD uptake into lymphocytes by the interaction in the lymphocytes interior. Previous studies have found that GSH can weaken MC-LR toxicity through conjugation [68–70]. Sipiä et al. [72] detected NOD-GSH in aquatic organisms. Thus, mediation of NOD-GSH conjugation by OPC may be the main mechanism through which OPC regulates hazardous NOD-induced oxidative toxicity in C. auratus lymphocytes. In the present study, the activity of GST significantly increased after fish lymphocytes were treated with OPC and the probability of GSH binding to NOD was enhanced. Best et al. [73] reported that decreases in GST activity inhibit the capacity of Danio rerio to detoxicate MC. Thus, GST may affect GSH conjugation with NOD in a manner similar to that in MC-LR [71]. Table 3 shows that C log P and HINK log P are reduced after MC-LR and NOD conjugation with GSH. Fliri et al. [74] stated that larger C log P values are accompanied by higher toxicity, i.e., lipophilic molecules tend to be multiple targets and are more toxic. Additionally, log P is not influenced by pH. In the present study, log P is more suitable than log D for evaluating the toxicity of NOD, MC-LR, and their conjugates. Lipophilicity, which may be reflected by C log P and HINK log P, is an important index that describes the toxicity of organic pollutants. Research by Kajiya et al. [75] indicated that cytotoxicity is attributable to the enhancement of lipophilicity. The partition coefficient in octanol/water decreased with decreasing transmembrane capability of NOD and MC-LR. Comparing reduced values of MCLR, MCLR-GSH, NOD, and NOD-GSH, decreases in the NOD group appeared to be more significant than decreases in the MC group. Lipophilicity, a physicochemical feature of a compound, depends on various physical and chemical characteristics, such as molecular surface area, molecular volume, and polarity [76]. Table 3 shows that PSA and the volumes of MC-LR or NOD increased after binding with GSH, regardless of the CPSA or MOLPROP descriptor calculations. Moreover, the ratios of PSA to molecular surface area and PV to molecular volume increased with increasing MCLR-GSH or NOD-GSH. Increases in NOD PV ratio were also more significant than increases in MC-LR PV ratio. Higher polarity usually can result in the easier metabolize. Therefore, NOD toxicity can decrease via binding with GSH. In the present experiment, we incubated fish lymphocytes with 100 ␮g/L NOD and different concentrations of OPC. OPC reduced the risk of NOD-induced intracellular toxicity by modulating oxidative stress indicators (e.g., decreasing ROS, increasing GSH content, activating SOD, CAT, GST, GPx, and GR) and proapoptotic proteins (e.g., upregulating bcl-2, downregulating bax expression), reducing intracellular Ca2+ , deactivating caspase-3 expression, and changing the structure of NOD. We also found that 1000 ␮g/L OPC is a suitable concentration for reducing NOD-induced toxicity. OPC concentrations higher than 1000 ␮g/L could lead to suppressive effects on the metabolic activity and cytokine expression of normal splenic lymphocytes [77]. Herfindal et al. [78] previously showed that addition of phenylglyoxal moieties to arginine can increase the log D value

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of NOD. In this research, calculated C log P and HINK log P values of NOD decreased after conjugation of NOD and GSH. NOD toxicity can be reduced by conjugation with GSH, which changes the physicochemical characteristics of NOD and increases its metabolism in the urine of organisms. In summary, OPC can effectively inhibit NOD-induced apoptosis of lymphocytes through various means, ultimately reducing risks of NOD-induced apoptosis. 5. Conclusion Utilization of the antioxidant OPC reduces hazardous NODinduced toxicity in lymphocytes. This research determined that 1000 ␮g/L OPC is an effective dosage for alleviating NOD-induced apoptosis of lymphocytes and that OPC affects mitosis by regulating antioxidant enzymes and deactivating proapoptotic proteins. Additionally, calculation results clearly illustrated that OPC-stimulated GSH and the conjugation of NOD and GSH can lower the lipophilicity and polarity of NOD. OPC, as a protective agent, can alleviate NOD-induced toxicity in C. auratus lymphocytes by regulating oxidative stress and inducing NOD-GSH detoxification. Further study is necessary to determine the exact OPC dosage required to protect fish immune systems from NOD-induced toxicity. The results of such a study will provide a more comprehensive understanding of both OPC effects and NOD toxicity. Acknowledgments This work was supported by the Natural Science Foundation of Zhejiang Province (Y5090190), the Program for Excellent Young Teachers in Hangzhou Normal University (JTAS 2011-01-012) and the ‘131’ Program for talents in Hangzhou City. References [1] F. Enzo, T. Emanuela, Human health risk assessment related to cyanotoxins exposure, Crit. Rev. Toxicol. 38 (2008) 97–125. [2] L. Pearson, T. Mihali, M. Moffitt, R. Kellmann, B. Neilan, On the chemistry, toxicology and genetics of the cyanobacterial toxins, microcystin, nodularin, saxitoxin and cylindrospermopsin, Mar. Drugs 8 (2010) 1650–1680. [3] W.W. Carmichael, Assessment of Blue-Green Algal Toxins in Raw and Finished Drinking Water, American Water Works Association Research Foundation, Denver, 2001. [4] K.L. Rinehart, K. Harada, M. Namikoshi, C. Chen, C.A. Harvis, M.H.G. Munro, J.W. Blunt, P.E. Mulligan, V.R. Beasley, A.M. Dahlem, W.W. Carmicheal, Nodularin, microcystin, and the configuration of Adda, J. Am. Chem. Soc. 110 (1988) 8557–8558. [5] M. Welker, H. von Döhren, Cyanobacterial peptides-nature’s own combinatorial biosynthesis, FEMS Microbiol. Rev. 30 (2006) 530–563. [6] D.P. Botes, P.L. Wessels, H. Kruger, M.T.C. Runnegar, S. Santikarn, R.J. Smith, J.C.J. Barna, D.H. Williams, Structural studies on cyanoginosins-LR, -YR, -YA, and -YM, peptide toxins from Microcystis aeruginosa, J. Chem. Soc. 1 (1985) 2747–2748. [7] R.E. Honkanen, M. Dukelow, J. Zwiller, R.E. Moore, B.S. Khatra, A.L. Boynton, Cyanobacterial nodularin is a potent inhibitor of type 1 and type 2A protein phosphatases, Mol. Pharmacol. 40 (1991) 577–583. [8] B.M. Gulledge, J.B. Aggen, A.R. Chamberlin, Linearized and truncated microcystin analogues as inhibitors of protein phosphatases 1 and 2A, Bioorg. Med. Chem. Lett. 13 (2003) 2903–2906. [9] B.M. Gulledge, J.B. Aggen, H. Eng, K. Sweimeh, A.R. Chamberlin, Microcystin analogues comprised only of Adda and a single additional amino acid retain moderate activity as PP1/PP2A inhibitors, Med. Chem. Lett. 13 (2003) 2907–2911. [10] D. Algermissen, R. Mischke, F. Seehusen, J. Göbel, A. Beineke, Lymphoid depletion in two dogs with nodularin intoxication, Vet. Rec. 169 (2011) 15. [11] H. Zhang, D. Shao, Y. Wu, C. Cai, C. Hu, X. Shou, B. Dai, B. Ye, M. Wang, X. Jia, Apoptotic response of Carassius auratus lymphocytes to nodularin exposure in vitro, Fish Shellfish Immunol. 33 (2012) 1229–1237. [12] W.X. Ding, C.N. Ong, Role of oxidative stress and mitochondrial changes in cyanobacteria-induced apoptosis and hepatotoxicity, FEMS Microbiol. Lett. 220 (2003) 1–7. [13] A. Jos, S. Pichardo, A.I. Prieto, G. Repetto, C.M. Väzquez, I.M. Moreno, A.M. Cameän, Toxic cyanobacterial cells containing microcystins induce oxidative stress in exposed tilapia (Oreochromis sp.) under laboratory conditions, Aquat. Toxicol. 72 (2005) 261–271.

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