Proteomic analysis of cadmium exposure in cultured lung epithelial cells: evidence for oxidative stress-induced cytotoxicity

Abstract

Human exposures to cadmium (Cd) compounds are common in the living environment. Cd is toxic, yet, little is known about its effect at the lung cell proteome level. Here, we provide a proteomic analysis of lung epithelial cells (LECs) treated with CdCl₂, with the aim of identifying protein response to Cd toxicity. Comparative proteome analysis was conducted to identify global changes in the protein expression profiles of sham-exposed and Cd-treated cells. Proteins were separated by two-dimensional electrophoresis and visualized by silver staining. We reported that while a low level (2 μM) of Cd treatment elicited negligible cytotoxicity and produced no significant proteome changes between the treated group and the control, however, a high level (20 μM) of Cd treatment induced obvious proteome changes and cell death in LECs. Differentially-expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. The proteins that were significantly up-regulated included heat-shock proteins (HSPs) and antioxidative stress proteins. Pretreatment with the thiol antioxidant glutathione before Cd treatment effectively abrogated the induction of these proteins and prevented cell death. Our results demonstrate that Cd causes oxidative stress-induced cell death, and these differentially-expressed proteins are defense proteins important for fighting against the Cd toxicity, while a low level of Cd may exert a more noticeable effect after long-term exposure, but not after transient exposure.

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Introduction

Cadmium (Cd) and Cd derivatives are ubiquitously-distributed in the living environment. In particular, the contamination of Cd in the food chain, occupational exposure and tobacco smoke provide important sources of human exposure to Cd.¹ Cd is toxic, and is ranked sixth out of the top ten hazardous substances.² It has been known for decades that Cd exposure can cause a variety of adverse health effects, including kidney dysfunction, lung diseases, disturbed calcium metabolism and bone effects.¹

In 1993, Cd and its derivatives have been classified as human carcinogens by the International Agency for Research on Cancer.³ The most obvious correlation between Cd and human diseases is found in the lungs.^1,4,5 The mechanism has, however, not been well-established. Evidence has indicated that ROS may be involved in Cd toxicity and carcinogenicity.⁶ Oxidative stress arises when ROS are produced faster than their removal by the cellular defense mechanisms, which can elicit a broad spectrum of responses depending on the level and the duration of exposure. In general, low levels of ROS are mitogenic and promote cell proliferation, while intermediate levels cause transient or permanent cell cycle arrest. High levels of ROS are detrimental and induced cell apoptosis or necrosis.^7,8

Inside cells, Cd induces the generation of abnormal or denatured proteins by reacting with vicinal thiols or by substituting for zinc in proteins. This has been recognized as the signal for the induction of HSPs.⁹ It has been demonstrated that exposure to Cd results in the induction of genes for metallothionein (MT), γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST) and elevated synthesis of glutathione (GSH), resulting in rapid and efficient detoxification of Cd ions as well as the ROS generated.^10,11 On the other hand, unfortunately, the activity of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase is suppressed by Cd,^12,13, which can explain why Cd induces oxidative stress, lipid peroxidation, and the associated toxicity.

In this study, we investigate the transient effects of Cd in lung epithelial cells (LECs), using a proteomic approach. Proteomics is a powerful tool developed to enhance our study of complex biological systems.¹⁴ This technique has been extensively applied to investigating the proteome response of cells to drugs and other diseases.¹⁵ However, to our knowledge, proteomic studies of Cd response on lung cells are lacking. Although two proteomic papers reported the Cd effects on human cells, the cell lines used are cancer cell lines [cervical cancer (HeLa) or leukemia (U937)],^16,17 making it hard to reveal/understand the proteomic response of Cd in normal cells. By using comparative proteome analysis between LECs and LECs treated with Cd, differentially-expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. We show that treatment with a high concentration of Cd induced the expression of a group of heat shock and antioxidative stress proteins, which served to protect the cells against oxidative stress-induced apoptosis exerted by Cd. On the other hand, a low level of Cd treatment produced no significant changes between the proteome profiles of the treated group and the control. These findings imply that a high level of Cd leads to oxidative stress-induced apoptosis, while a low level of Cd may exert more noticeable effect after long-term exposure, but not after transient exposure.

Results

Cytotoxicity of Cd in LECs

Since different types of cell lines would have different sensitivity to Cd exposure, to compare the sensitivity of LECs to Cd, cells were treated with varying concentrations of CdCl₂ and cell viability was determined by the NBB assay. Increasing Cd concentrations exhibited cytotoxicity to LECs. Cell viability is unaffected at 1 or 2 μM CdCl₂ but severely compromised at higher concentrations of CdCl₂. From the data, the LC₅₀ was determined to be approximately 20 μM (Fig. 1).

Fig. 1 Dose-dependent cytotoxicity of Cd on LECs. Cells were plated in 96-well plates at 2 × 10⁴ per well and incubated overnight. On the next day, cells were dosed with CdCl₂. After 24 h, cell viability was measured by NBB staining assay. The percentage of viability was plotted as 100% for control (no treatment of Cd). Results are expressed as mean ± S.D. of triplicate samples and reproducibility was confirmed in three separate experiments. ^*A significant difference (P < 0.05) as compared with control. The results are representative of three independent experiments.

Comparison of proteome profiles between LECs and LECs treated with different dosages of Cd

From the cytotoxicity results, we therefore wish to examine the proteome changes of LECs dosed with low and high concentrations of Cd. After treatment, LEC cell extracts were isolated and applied to 2-DE and proteins visualized by silver staining. 2D gels were run three times for each sample to minimize gel-to-gel variation. Fig. 2 shows the representative gel images for normal control and LECs treated with a non-cytotoxic level (2 μM) of Cd for 24 h. Overall, the two gels displayed a very similar pattern. More than 1000 spots were detected in a gel ranging from 6.5 to 200 kDa of molecular mass with pIs between 3 and 10. Spot volume comparison was made between the samples with the ImageMaster program. There were no significant differences of protein spots in LECs treated with 2 μM Cd for 24 h (ESI Table S1†). We also examined the proteome profiles of LECs treated with 2 μM Cd at 2, 4, 6, 8 and 12 h time points, again, no significant differences were found as compared to control (data not shown).

Fig. 2 Time course experiment on LECs treated with 2 μM Cd for 24 h showed no significant changes in proteome profile compared with control. Cells were treated with 2 μM Cd and harvested at 0, 2, 4, 6, 8, 12 and 24 h. They were then lysed and subjected to 2-DE and image analyses. Only data at time 0 h (left) and 24 h (right) were shown. The results are representative of three independent experiments.

Fig. 3 shows the representative gel images of normal control and LECs treated with a high level of (20 μM) Cd for 24 h. In contrast to 2 μM Cd, 20 μM Cd treatment produced a significant difference of spots between the two gels, indicating their potential roles as primary Cd-responsive proteins induced by Cd. Fig. 3B is a comparative montage view of the selected regions shown in Fig. 3A, where significant differences in protein expression level were marked. These are the spots in which protein identity was later confirmed by MS analysis and selected for further study in this work (ESI Table S1†).

Fig. 3 Representative gel images of LECs treated with or without 20 μM Cd for 24 h visualized by two-dimensional gel (12.5%) and silver staining. A: Control LECs and LECs treated with 20 μM Cd. B: Comparative montage view of the regions shown in A, where significant differences in protein expression level were marked. Differentially-expressed proteins are numbered from 1 to 7 and indicated by arrows. The protein spots were excised, in-gel-digested with trypsin and identified by MALDI-TOF-MS analysis. The results are representative of three independent experiments.

Protein identification

Treatment with a high level of Cd altered the protein profile in LECs (Fig. 3). Protein spots that displayed significant differences (spots over 2-fold differences) were cut out and subjected to trypsin digestion, MALDI-TOF mass spectra measurement and database searching. Table 1 summarizes the identified proteins and their alterations between normal control and 20 μM Cd treated cells. We are interested in seven spots which comprise HSPs (heat-shock proteins), including HSP70, HO-1 (heme oxygenase-1; HSP32), HSP27 and αB-C (αB-crystallin), and antioxidative stress proteins such as AR (aldose reductase) and FLC (ferritin light chain). Spots 6 and 7 were both αB-C, but spot 6 was the phosphorylated form of αB-C. This was evidenced by the tryptic peptide mass spectrum for differences in peptide masses of 0.08 kDa that were not found in spot 7 (results not shown). The identity and expression levels of these proteins in basal and Cd-treated cells were further confirmed by Western blot analysis, which showed a similar trend of induction as appeared in 2-D gels (Fig. 4).

Fig. 4 Confirmation of the MS-identified proteins by Western blot analysis. LECs were treated with 20 μM Cd for 24 h. Total cellular proteins were subjected to Western blot analysis for the detection of HSP70, AR, HO-1, HSP27, FLC, and αB-C. The same blot was also reprobed with β-actin to ensure equal loading. The results are representative of three independent experiments.

Table 1 Identification of differentially-expressed proteins between LECs and LECs treated with 20 μM of CdCl₂ for 24 h

Spot no.	Identified protein	NCBI accession no.	Coverage^a (%)	Mass (kDa)/pI	Volume (%) (mean ± S.D.)		Fold difference^b	Cellular function
					Untreated	Treated
a Sequence coverage (%) of full length protein at 25 ppm. b Average expression level in Cd-treated cells compared to LECs from 3 independent analyses (+, increase). c ND, non-detectable. d N/A, not applicable.
1.	Heat-shock protein 70	27704462	44	70.2/5.6	ND^c	0.737 ± 0.091	N/A^d	Chaperone
2.	Aldose reductase	27465603	36	36.2/7.1	0.165 ± 0.00293	0.612 ± 0.052	+3.7	Glucose reduction
3.	Heme oxygenase-1	6981032	42	33/6.1	ND	0.783 ± 0.081	N/A	Heme cleavage
4.	Heat-shock protein 27	1170367	46	22.9/6.1	0.081 ± 0.00357	0.981 ± 0.046	+12.1	Chaperone
5.	Ferritin light chain	2119695	32	20.8/6.0	0.021 ± 0.00970	0.171 ± 0.0168	+8.1	Intracellular iron regulation
6.	αB-crystallin (phosphorylated)	16905067	49	20/6.8	ND	0.780 ± 0.037	N/A	Chaperone
7.	αB-crystallin	16905067	51	20/6.8	0.350 ± 0.014	1.117 ± 0.069	+3.2	Chaperone

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Induction of stress proteins by Cd is modulated by intracellular GSH in LECs

Since intracellular GSH level is vital for the redox homeostasis of cells and it has been shown that GSH is a first line of defense against Cd toxicity,¹⁸ we investigated the protective role of intracellular GSH in the induction of stress proteins by Cd in LECs, we tested the effect of pretreatment with GSH. GSH was added 1 h before the addition of CdCl₂. Pretreatment with 20 mM GSH before Cd treatment effectively inhibited the induction of stress proteins and sustained a similar proteome profile to that of control cells (Fig. 5).

Fig. 5 Induction of stress proteins by Cd is inhibited by antioxidant GSH. LECs were exposed to CdCl₂ (20 μM) in the absence or presence of 20 mM GSH (pH adjusted to 7.6). GSH was added 1 h before the addition of CdCl₂. LECs were also treated with GSH alone. After treatment for 24 h, cells were lysed and whole cell lysate was prepared. Effects of GSH on protein expression profiles of six proteins in basal and Cd-treated cells were assessed by 2-DE analyses and shown in montage view. The results are representative of three independent experiments.

Cd-induced cell death is correlated with oxidative stress

To further confirm that Cd-induced cell death in LECs is due to oxidative stress, LECs were exposed to 20 μM CdCl₂ for 24 h in the absence or presence of GSH (20 mM). GSH was added 1 h before the addition of CdCl₂. Western blot analyses of pro-apoptotic protein Bax and procaspase-3 levels (Fig. 6A) showed that pretreatment with 20 mM of GSH effectively suppressed Bax expression and procaspase-3 activation in LECs treated with 20 μM Cd. Moreover, nuclear staining using DAPI showed that pretreatment with GSH before Cd treatment effectively protected the cells against oxidative stress-induced apoptosis by Cd. In the absence of GSH pretreatment, massive cell death can be observed by Cd treatment (Fig. 6B), indicating the important role of oxidative stress in Cd-induced cell death.

Fig. 6 Cd-induced cell death is correlated with oxidative stress and is countered by GSH pretreatment. LECs were exposed to 20 μM CdCl₂ for 24 h in the absence or presence of GSH (20 mM). A: Western-blot analyses for the detection of Bax and procaspase 3, using Bax and procaspase 3 antibodies respectively and monoclonal β-actin antibody to monitor the loading difference. B: The corresponding assay for the determination of apoptotic cells. The percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells counted, multiplied by 100. Black bars indicate Cd treatment. Results are expressed as mean ± S.D. of triplicate samples and reproducibility was confirmed in three separate experiments. ^*A significant difference (P < 0.05) as compared with cells treated with Cd alone. The results are representative of three independent experiments.

Discussion

Our previous study reported the characterization of Cd exposure in LECs and showed that treatment of LECs with apoptotic concentrations of Cd up-regulates the expression of oxidant stress genes (MT, GST and γ-GCS), enhances the DNA binding activities of redox sensitive transcription factors (AP-1 and NF-κB), and alters GSH homeostasis, which are important for fighting against the cytotoxicity during the course of oxidative stress-induced apoptosis exerted by Cd.¹¹ In this study, we further study them using a proteomic approach.

It is documented that Cd alters immediate early gene expressions and activates various signaling pathways.^19–23 In addition, experiments using cDNA microarrays have demonstrated that treatment of cells with low levels of Cd altered their gene expression profile.²⁴ However, we could not detect any significant changes in protein expression when cells were treated with 2 μM Cd for 24 h. This was probably because changes in gene expression level do not necessarily correlate to alterations in protein expression level. Most kinase signaling pathways are regulated rapidly by post-translational modifications, such as phosphorylation of target proteins and effectors. It may be that 2-DE wasn't sensitive enough to pick up minor changes induced by low levels of Cd. In addition, we cannot also rule out the possibility that different cell lines may respond differently to the treatment of Cd. Furthermore, while a low level of Cd exposure has been shown to induce cell transformation, this occurred after long-term exposure.²³ It is believed that chronic low levels of Cd exposure to cells induce sustained oxidative stress and may gradually promote the carcinogenesis process over a more prolonged period of time.^22,23 It is therefore not surprising that our proteomic results were unable to detect significant proteome changes in LECs treated with 2 μM Cd for a relatively short period of time.

On the other hand, we were able to identify major proteins that are involved in the cellular response to a high level of Cd exposure. Comparative analysis of proteome profiles between the parental and Cd-treated cells allowed the identification of proteins whose levels were altered upon 20 μM Cd treatment, identifying them as primary defense proteins against Cd. Our results also demonstrated that Cd-induced cell death in LECs is mainly due to oxidative stress, as pretreatment with thiol antioxidant GSH before Cd treatment effectively abrogated the induction of stress proteins and protected the cells against oxidative stress-induced apoptosis by Cd.

The up-regulation of HSP stress proteins by the cells after 20 μM Cd treatment is a cellular-protective response. HSPs are a class of evolutionary conserved proteins with various molecular sizes and diverse functions among different species. Basically, they are both constitutively-expressed and inducible in the cells.²⁵ The importance of HSPs in living organisms has been demonstrated since the inception of HSP gene cloning. To date, seven families of HSP have been identified. Based on the recent suggested guidelines for nomenclature of the heat-shock proteins, they are classified as the HSP families; HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), HSPB (small HSP) as well as the chaperonin families; HSPD/E (HSP60/HSP10) and CCT (TRiC).²⁶ In the present study, we demonstrated the induction of the HSP70 and small HSP members among the stress proteins induced by 20 μM Cd.

HSP70 is part of the HSPA (HSP70) family, whose members act as molecular chaperones and are involved in many cellular functions such as protein folding, transport, maturation and degradation. They recognize and bind to nascent polypeptide chains as well as partially folded intermediates of proteins to prevent their aggregation and misfolding. The HSP70 family has also been shown to inhibit cytochrome c release and to suppress the activity of caspases and thereby counteract the apoptotic cascades.^27,28

HSP27 and αB-C belong to the HSPB (small HSP) family. They are expressed constitutively in cells, and their expression is increased in response to various types of stress, including heat shock, drugs and oxidants. They exist mainly as oligomers, which display chaperone-like activity, serving as a site where unfolding proteins may bind and refold. The refolding process is ATP- and HSP70-dependent and protects the cell against oxidative stress and apoptosis. Under stress conditions, these large oligomers rapidly dissociate into small oligomers as a result of phosphorylation by kinases.²⁹ HSP27 has been shown to protect against apoptosis by regulating the activation of the phosphoinositide 3-kinase/protein kinase B pathway³⁰ and to inhibit cytochrome c-dependent activation of procaspase 9.³¹ αB-C has also been shown to be a negative regulator of apoptosis by inhibiting the autocatalytic maturation of caspase 3.³² In the present study, the up-regulation of HSP70, HSP27 and αB-C appeared to assist in chaperoning proteins unfolded or denatured by Cd, and antagonizing the cell against oxidative stress and apoptotic cascades triggered by Cd. Our data is in agreement with other studies that HSP70, HSP27 and αB-C are induced by Cd in human lens epithelial cells and hepatic-derived cells.^33,34

An increase in ROS levels by Cd can perturb the cell redox status, thereby producing damage to DNA, proteins and lipids, and eventually cell death. Therefore, the cells try to adjust the intracellular environment to maintain the state of redox balance. HO-1 and FLC were up-regulated significantly after the cells were treated with 20 μM Cd. HO-1 is a microsomal enzyme that cleaves heme to produce biliverdin, inorganic iron and carbon monoxide.³⁵ HO-1's activity is highly inducible in response to numerous stimuli, including heme, heavy metals and hormones. Increased levels of HO-1 can be used as an indication of exposure to oxidative stress.³⁶ Growing evidence shows that HO-1 can exert antiproliferative and antiapoptotic effects and participate in general cellular defense mechanism against oxidative stress in mammalian cells.^37,38 Ferritins regulate iron metabolism. Mammalian ferritins consist of two types of polypeptide chains (ferritin heavy chains and FLCs).³⁹ The most prominent role of mammalian ferritins is to provide iron-buffering capacity in the cells. And it has been shown that ferritin is able to bind with divalent metal ions including Cd.⁴⁰ To examine oxidative stress, Cairo et al.⁴¹ showed that ROS induces a 6-fold increase in the rate of ferritin synthesis in rat liver. Similarly, our results show that 20 μM Cd induced an ∼8-fold increase in the FLC protein level. Obviously, the cleavage of heme by HO-1 as well as the binding of Cd with ferritin produced an increase in intracellular free iron levels. The increase in the cellular iron pool induces ferritin gene transcription in an attempt to limit iron bioavailability. Our data is in agreement with other studies that HO-1 is induced by Cd treatment.^42,43 Meanwhile, AR, a member of the aldoketoreductase family, has been shown as a novel antioxidative stress protein under oxidative stress conditions by metabolizing several aldehyde products including 4-hydroxy trans-2-nonenal, a major toxic product of lipid peroxidation as a result of oxidative stress.⁴⁴ Therefore, the up-regulation of AR is expected after 20 μM Cd treatment, to protect the cell against oxidative stress. From the above, we can see that all these up-regulated HSPs and antioxidative stress proteins fight against the cytotoxicity exerted by Cd.

In summary, our proteomic study demonstrated that a high level (20 μM) of Cd causes oxidative stress-induced apoptosis in lung cells. This is supported by the fact that pretreatment with antioxidant 1 h before Cd treatment effectively prevented cell death and the induction of HSPs. Western blot analyses demonstrated that pretreatment with antioxidant effectively suppressed Bax expression and procaspase 3 activation in LECs treated with 20 μM Cd. The exposure of lung cells to 20 μM Cd caused oxidative stress and subsequently up-regulated stress proteins, which served to protect the cells from the Cd cytotoxicity. By contrast, a low level (2 μM) of Cd produced no significant changes on the proteome profile and may exert more noticeable effect after long-term exposure, but not after transient exposure. Further studies are underway to determine the effect of long-term Cd exposure on these cells and will hopefully provide more insights into this area in the future.

Materials and methods

Materials

Cadmium chloride (CdCl₂) and glutathione (GSH) were purchased from Sigma (St. Louis, MO). PlusOne 2-D Clean-Up kit and Silver Staining kit were purchased from GE Healthcare (Uppsala, Sweden). All other general chemicals were purchased from GE Healthcare and Sigma. Antibodies used for Western blot were purchased from Sigma, Upstate Biotechnology (Lake Placid, NY), StressGen Biotechnologies (Victoria, BC, Canada) and Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture

A rat lung epithelial cell line (LEC) was isolated and characterized as previously described.⁴⁵ The cells are considered to be the stem cells of the type II alveolar epithelium. Cells were routinely grown in F-12 complete media containing 10% (v/v) newborn calf serum, 100 units ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, and 2 mM glutamine at 37 °C in an atmosphere of 5% CO₂/95% air. All tissue culture reagents were obtained from Gibco-BRL (Grand Island, NY).

Cd treatment

Cells were grown to 75% confluence and then were either sham-exposed or treated with different concentrations of CdCl₂. Cells were pretreated with GSH for 1 h before the addition of Cd. Cell viability was measured by naphthol blue black (NBB) staining assay as described previously.⁴⁶

Quantification of apoptotic death

To detect apoptosis, nuclear staining was performed using 1 μg ml⁻¹ DAPI, and cells were analyzed with a fluorescence microscope. Apoptotic cells were identified by morphology and by condensation and fragmentation of their nuclei. The percentage of apoptotic cells was calculated as the ratio of apoptotic cells to total cells counted, multiplied by 100. Three separate experiments were conducted and at least 300 cells were counted for each experiment.

Cell lysate preparation and conditions of Western blot and two-dimensional PAGE

After treatment, cells were then washed thrice with ice-cold PBS, scraped into a centrifuge tube, and then harvested by centrifugation at 1000 g for 5 min at 4 °C. For Western blot analysis, cell pellets were lysed in radioimmunoprecipitation assay buffer according to the protocol described by Upstate Biotechnology. Equal amounts of proteins (40 μg) were fractionated on a SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20 and probed with various primary antibodies. After incubation with secondary antibodies, immunoblots were visualized with the enhanced chemiluminescence detection kit (GE Healthcare). For two-dimensional PAGE analysis, cell pellets were lysed in lysis buffer [8 mol l⁻¹ urea, 4% (w/v) CHAPS], incubated on ice for 30 min, and centrifuged at 16 000 g for 5 min at 4 °C. The supernatant was saved and then further purified by using the PlusOne 2-D Clean-Up kit in accordance with the manufacturer. The purified samples were finally redissolved in rehydration buffer (8 mol l⁻¹ urea, 2% CHAPS), aliquoted into several tubes, and stored at −80 °C after protein quantitation. Two-dimensional PAGE was done on 80 μg of cleaned-up cell extract with IPGphor IEF (GE Healthcare) and Hoefer SE 600 electrophoresis units. All gels were visualized by silver staining using the PlusOne Silver Staining kit in accordance with the manufacturer.

Image analysis, MALDI-TOF-MS analysis, and protein identification

The stained gels were scanned using an ImageScanner (GE Healthcare) operated by the LabScan 3.00 software. Image analysis was carried out by using the ImageMaster 2D Elite software 4.01. Only spots (changed in expression for more than 2-fold) or spots that either appeared/disappeared were selected for analysis with MS. Protein spots were excised and transferred into siliconized 1.5 ml Eppendorf tubes. Gel chips were destained, dehydrated with acetonitrile, and then rehydrated in trypsin solution (10 μg ml⁻¹ in 25 mmol l⁻¹ NH₄HCO₃) at 37 °C overnight. The digest was then applied onto a sample plate and coated with matrix (α-cyano-4-hydroxycinnamic acid). Tryptic peptide MS were obtained using a Voyager-DE STR MALDI-TOF-MS (Applied Biosystems, Foster City, CA). Protein identification was performed by searching the NCBInr protein database using MS-Fit. The criteria for searching were set with 25 ppm or better mass accuracy, at least four matching peptide masses, and molecular weight and isoelectric point (pI) matching estimated values from gels. Species search was limited to Rattus norvegicus.

Statistical analysis

Statistical analysis was performed by using two-tailed Student's t test, and P < 0.05 was considered significant. Data are expressed as the mean ± SD of triplicate samples, and the reproducibility was confirmed in three separate experiments.

Acknowledgements

This work was supported by National Natural Science Foundation of China Grants 31170785 and 81101785 (Andy T. Y. Lau), Fund for University Talents of Guangdong Province (Andy T. Y. Lau), and Guangdong Natural Science Foundation of China Grant S2012030006289. We would like to thank members of the Lau And Xu laboratory for critical reading of this manuscript.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3tx50014d

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