The authors have declared that no competing interests exist.
Conceived and designed the experiments: YB YC TW CJH YS. Performed the experiments: WW YH BL DWS AAR GY RL. Analyzed the data: WW DWS AAR YS YB. Contributed reagents/materials/analysis tools: CJH YC YS YB. Wrote the paper: WW DWS TW CJH AAR YC YS YB.
The potential cytotoxicity of cadmium selenide (CdSe) quantum dots (QDs) presents a barrier to their use in biomedical imaging or as diagnostic and therapeutic agents. Sulforaphane (SFN) is a chemoprotective compound derived from cruciferous vegetables which can up-regulate antioxidant enzymes and induce apoptosis and autophagy. This study reports the effects of SFN on CdSe QD-induced cytotoxicity in immortalised human hepatocytes and in the livers of mice. CdSe QDs induced dose-dependent cell death in hepatocytes with an IC50 = 20.4 μM. Pre-treatment with SFN (5 μM) increased cell viability in response to CdSe QDs (20 μM) from 49.5 to 89.3%. SFN induced a pro-oxidant effect characterized by depletion of intracellular reduced glutathione during short term exposure (3–6 h), followed by up-regulation of antioxidant enzymes and glutathione levels at 24 h. SFN also caused Nrf2 translocation into the nucleus, up-regulation of antioxidant enzymes and autophagy. siRNA knockdown of Nrf2 suggests that the Nrf2 pathway plays a role in the protection against CdSe QD-induced cell death. Wortmannin inhibition of SFN-induced autophagy significantly suppressed the protective effect of SFN on CdSe QD-induced cell death. Moreover, the role of autophagy in SFN protection against CdSe QD-induced cell death was confirmed using mouse embryonic fibroblasts lacking ATG5. CdSe QDs caused significant liver damage in mice, and this was decreased by SFN treatment. In conclusion, SFN attenuated the cytotoxicity of CdSe QDs in both human hepatocytes and in the mouse liver, and this protection was associated with the induction of Nrf2 pathway and autophagy.
Synthesis of nanoparticles (NPs) has become increasingly common, with some NPs now being produced commercially, including cadmium selenium (CdSe) quantum dots (QDs) [
Chemoprevention with natural compounds represents an attractive approach to increase cellular defence against environmental and endogenous insults [
CdSe (10:1) QDs showed notable cytotoxicity in HHL-5 cells after 12 h exposure. The cytotoxicity was more significant after 24 h with an IC50 = 20.4 μM CdSe pairs which is equivalent to 0.78 nmol core QDs/ml. However, when the cells were pre-treated with 5 μM SFN for 24 h, the cytotoxicity induced by 20 μM CdSe QDs (24 h exposure) significantly decreased, raising cell viability from 49.5 to 89.3% (P<0.01,
Effect of SFN pre-treatment on CdSe QD-induced cytotoxicity. HHL-5 cells were pre-treated with SFN (5 μM) for 24 h, and then incubated with 20–30 μM CdSe QDs for another 24 h. Cytotoxicity was measured by MTT assay, and data shown as means ± SD (n = 6). **P<0.01.
(A) Pre-treatment with 5 μM SFN abrogated the protective effect with an observable increase the viable cell percentage (double negative, Q1) relative to the non-pre-treated cells. Samples were run on a BD Accuri C6 flow cytometer (Ex 488nm; Em 533/30nm BP & 670nm LP). Data is representative of at least 3 experiments. (B) Effect of SFN on CdSe QD-induced apoptosis and necrosis. Apoptosis and necrosis were determined by flow cytometry using Annexin V/PI staining. *P<0.05, **P<0.01.
GSH is the most important and abundant endogenous antioxidant in mammals and its regulation represents an important research topic in chemoprevention [
(A) Effect of SFN on intracellular GSH levels. HHL-5 cells were exposed to SFN (2.5, 5 and 10 μM) with DMSO (0.1%) as a control for 0, 3, 6, 12 and 24 h. The levels of intracellular GSH were measured by an HPLC assay. Data are shown as means ± SEM (n = 3). (B) Effect of SFN and BSO pre-treatment on CdSe QD-induced cytotoxicity. HHL-5 cells were pre-treated with SFN (5 μM) and/or BSO (100 μM) for 24 h, and then incubated with 20 μM CdSe QDs for another 24 h. Cytotoxicity was measured by MTT assay, and data shown as means ± SD (n = 6). *P<0.05, **P<0.01.
The depletion of intracellular GSH is essential for SFN to facilitate the modification of Cys residues in Keap1. This enables Nrf2 to escape Keap1-dependent ubiquitination and degradation, and results in activation of Nrf2 [
TR-1 is driven by the Keap1-Nrf2-ARE signalling pathway. CdSe QDs (20 μM) decreased HHL-5 cell viability to 25.4% without SFN pretreatment. siTR-1 was found to have no significant effect on cell viability. However, siNrf2 knockdown indicated that diminished Nrf2 signalling enhanced the cytotoxicity of CdSe QDs, i.e. cell viability decreased from 25.4 to 19.7% (P<0.05) (
TR-1, Keap1 or Nrf2 were knocked down, respectively. Allstars (AS) were used as a negative control. Cells were incubated with 5 μM SFN or DMSO (0.1%) control for 24 h then exposed to 20 μM CdSe QDs for 24 h. Cell viability was measured by MTT assay, and data shown as means ± SD (n = 6). Significant levels (*P<0.05; **P<0.01) in comparison with AllStars (AS) negative controls.
Metallothionein (MT) is a small-molecular weight, cysteine-rich protein that binds metals, and it is known that MT is a Nrf2–driven gene since there is at least one ARE in the MT regulatory region [
Deceased cellular GSH levels activate autophagy [
HHL-5 cells 48 h after seeding were (A) exposed to SFN for 6 or 24 h, or (B) treated with varying levels of CdSe QD for 6 or 24 h. The expression of LC-3-II was analysed by Western blot analysis.
Wortmannin acts as a selective inhibitor of type III PI-3K [
(A) HHL-5 cells were pre-incubated with wortmannin (0.1 μM) for 6 h, and then exposed to SFN (5 μM) for 24 h. There was a further 24 h exposure with 20 μM CdSe QDs. (B) MEF ATG5-/- cells were pre-incubated with SFN (5 μM) for 24 h and then exposed for a further 24 h with CdSe QDs (10–30 μM). Cytotoxicity was measured by MTT assay. Data are shown as means ± SD (n = 6) (**P<0.01).
Liver toxicity in mice was evaluated by histological examination after H&E staining (
Liver toxicity in mice was evaluated by histological examination after H&E staining. CdSe QDs caused hepatocellular necrosis with cell swelling and modulated nuclear morphology as indicated by the arrow. The original magnification was 400X.
SFN is an isothiocyanate derived from cruciferous vegetables such as broccoli and cauliflower and its anti-cancer activity was discovered 20 years ago [
Although the two antioxidant enzymes, TR-1 and QR-1, were shown to be less significant in the protection against CdSe QDs toxicity, the role of Nrf2 in protection was prominent. There are many other Nrf2-ARE driven antioxidant enzymes such as haem oxygenase-1 (HO-1), glutathione peroxidases (GPXs), GSTs, and peroxiredoxin, which may also be involved in the protection against oxidative stress [
In summary, SFN treatments provided protective effects on CdSe QD-induced cytotoxicity in human hepatocytes and the livers of the mice. The mechanisms of protection were mainly via activation of Nrf2-ARE and autophagy pathways that potentiate the protective effect of SFN against CdSe QD-induced cell death in hepatocytes.
SFN, 4-methylsulfinylbutyl isothiocyanate was purchased from Alexis Biochemicals (UK). Methylthiazolyldiphenyl-tetrazolium bromide (MTT), wortmannin, 3-methyladenin (3-MA), cadmium perchlorate, dimethyl selenourea, LC3 antibody and GenElute™ total mammalian RNA isolation kit were all purchased from Sigma-Aldrich (UK). Complete protease inhibitors were obtained from Roche Applied Science (UK). Rabbit polyclonal primary antibodies to Nrf2, TR-1, QR-1 goat polyclonal primary antibody to Keap1, β-actin, rabbit polyclonal primary antibody to Sam68 (Src-associated in mitosis 68 kDa, a RNA binding protein), HRP-conjugated goat anti-rabbit and rabbit anti-goat IgG were all purchased from Santa Cruz Biotechnology (Heidelberg, Germany). siRNAs for Nrf2, TR-1, QR-1, Keap1 and AllStars negative control were all purchased from Qiagen (West Sussex, UK). Annexin V-FITC apoptosis detection kit was purchased from eBioscience (UK). Electrophoresis and Western blotting supplies were obtained from Bio-Rad (UK), and the Chemiluminescence kit was from GE Healthcare (Little Chalfont, UK).
CdSe nanoparticles were synthesized by the microwave heating of an aqueous solution of cadmium perchlorate (CdCl2O8) as a source of cadmium ions with N,N-dimethyl selenourea (C3H8N2Se) as a source of selenium ions, in the presence of sodium citrate as a stabilizer [
Immortalised human hepatocytes (defined as HHL-5) were kindly supplied by Dr A. Patel, Medical Research Council (MRC) Virology Unit (Glasgow, UK). The primary hepatocytes were immortalised with Moloney’s mouse leukaemia virus and contained markers of hepatocyte and biliary phenotype, cytochrome P450 and albumin [
The MTT cell proliferation assay was used to detect the toxicity of CdSe QDs and the protective effect of SFN. HHL-5 cells were seeded in 96-well plates in DMEM with 10% FCS at a concentration of 0.5–1.0 × 104 cells in a final volume of 100 μl per well. SFN treatments and controls had an equal final concentration of DMSO (0.05%). After 24 h treatment with SFN, cells were exposed to different concentrations of CdSe QDs in fresh medium for a further 24 h period. After treatment, the medium was removed, 100 μl (5 mg/ml) MTT was added, and incubated at 37°C for 1 h to allow the MTT to metabolize. The formazan formed was then re-suspended in 100 μl DMSO per well. The final absorbance was recorded using a microplate reader (BMG Labtech Ltd, UK) at a wavelength of 550 nm and a reference wavelength of 650 nm.
HHL-5 cells were seeded on 96-well plates at density of 1×104/well in DMEM with 10% FCS. After 24 h, the cells were transfected with siRNA for TR-1, QR-1, Keap1, Nrf2 or Allstars (no homology to any known mammalian gene). Briefly, the cell culture medium was replaced with 100 μl medium containing12% FCS, then 10 nM siRNA with 0.6 μl Hiperfect transfection reagent (in 20 μl medium without serum and antibiotics) was incubated at room temperature for 10 min before being gently added drop-wise to the cells. AllStars was used as a negative control siRNA. After 24 h incubation with siRNAs, protective compound SFN was added in fresh medium for a further 24 h, then the effect of CdSe QDs (20 μM, 24 h) on cell death was measured using the MTT cell viability assay. The siRNA knockdown efficiency of Nrf2 and Keap1 was characterized using Western blot analysis (
For total protein, HHL-5 cells were washed twice with ice-cold PBS, harvested by scraping in 20 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Nonidet P40 (NP-40) containing mini-complete proteinase inhibitor and 1 mM PMSF and then incubated in an ice bath for 20 min and centrifuged at 12,000 g for 15 min at 4°C. Supernatant was collected and the protein concentration determined by the Brilliant Blue G dye-binding assay of Bradford using BSA as a standard. For the nuclear protein, the extraction was performed using a Nuclear Extract Kit (Active Motif, UK), following the manufacturer’s instructions. Protein extracts were heated at 95°C for 5 min in loading buffer and loaded onto 10% SDS-polyacrylamide gels together with a molecular weight marker. After routine electrophoresis and transfer, the PVDF membrane was blocked with 5% fat free milk in PBST (0.05% Tween 20) for 1 h and incubated with a specific primary antibody in 5% milk in PBST for 1 h. The membrane was washed three times for 45 min with PBST and then incubated with the secondary antibody diluted with 5% milk in PBST for 1 h. After further washing the membrane three times for 45 min with PBST, antibody binding was determined by a Chemiluminescence detection kit and densitometry was measured by Fluor Chem Imager (Alpha Innotech, San Leandro, CA).
Intracellular concentrations of reduced GSH were determined by HPLC analysis of cell lysates derivatised with monobromobimane (mBBr). The procedure was adapted from Newton and Fahey [
Total RNA was extracted from HHL-5 cells using GenElute™ total mammalian RNA kit (Sigma, UK) according to the manufacturer's instruction. First strand cDNA was synthesised with 1 μg of total RNA using qScript cDNA SuperMix (Quanta BioSciences, UK). MT-1A mRNA quantification was determined by TaqMan real-time PCR using the Roche LightCycler 480 System (Roche Diagnostics, Switzerland). Forward primer,
HHL-5 cells were seeded on 12-well plates at a density of 5×104 cells per well and incubated at 37°C for 48 h. After treatment with 5–10 μM SFN for 24 h, cells were exposed to 10–20 μM CdSe NPs for 6 and 24 h. Cells were then detached from the wells using trypsin and collected by centrifugation at 180 g for 6 min at 4°C, and the pellets washed with cold PBS before being re-suspended in 400 μl cold PBS. Flow cytometry was performed with a BD Accuri C6 Flow Cytometer using 488 nm excitation with 533/30 nm band pass (BP) and 670 nm long pass (LP) filtered detection. The effect of CdSe QD on apoptosis was assessed using an Annexin V-FITC apoptosis detection kit (eBioscience, UK), according to the manufacturer's instructions. Cells were trypsinised and collected, Annexin V-FITC (fluorescein isothiocyanate) was used to label the apoptotic cells and propidium iodide (PI) used to stain the necrotic cells. For each sample 10,000 events were collected and the data were analysed using FlowJo software (Treestar Inc. USA).
ICR mice were obtained from the experimental animal centre in Heilongjiang University of Chinese Medicine, and maintained in a Specific Pathogen Free animal house according to the guidelines of the Institutional Animal Care Use Committee. This study was approved by the Animal Experimental Ethics Committee, Heilongjiang University of Chinese Medicine, Harbin, China (License No: SCXK-Hei-2012016). Regarding the acute exposure to CdSe QDs, 36 mice (BW 20-23g) were randomized into control, CdSe, and SFN treatment + CdSe groups with 12 mice (6 male and 6 female) in each group. Mice in the CdSe group were administrated intraperitoneally with 0.2 ml 400 μM CdSe QDs once. During the first 4 hours after administration, mouse reactions such as twitch, secretions from the eyes and nose, respiration and heartbeat rate were monitored. Mice in the SFN protection group were given SFN (40 mg/kg BW) every other day for 14 days through oral gavage (three doses before CdSe injection and four after CdSe injection in every other day). Control mice were given PBS orally only. Mice were then sacrificed 24 h after the last gavage of SFN through peritoneal injection with 10% chloral hydrate (anaesthetic agent) solution (0.3 ml/100g BW). During the whole experiment, there were no animals died of liver damage. The liver tissues were fixed in 10% buffered formal saline for 48 h before processing according to the defined pathologic protocols [
Data are represented as the mean ± SD or SEM. The differences between the groups were examined using the one-way ANOVA test, or Student’s t-test. A
(A) A drop of QDs solution was cast onto a carbon film grid prior to the measurement using a JOEL 2000EX TEM with the accelerating voltage of 200 kV. (B) Histogram shows the size distribution of CdSe QDs obtained by measuring 100 QDs from different parts of the grid.
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(A) HHL-5 cells were treated with SFN for 24 h. (B) cells were pre-treated with SFN (5 μM) for 24 h then treated with CdSe for further 24 h. DMSO (0.1%) was used as a control. (C) siRNA knockdown Keap1 and Nrf2 in HHL-5 cells. Cells were seeded into 10cm dish. After 24 h, cells were treated with siKeap1 or siNrf2. Allstars (AS) was used as a negative control. After 24 h treatment, medium was changed and 5 μM SFN or DMSO (0.05%) was added for further 24 h. Nrf2 in nuclear extract and Keap1 in cytosol (D) were detected using Western blot analysis.
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After seeding for 48 h, cells were exposed to SFN for 24 h (A) or 48 h (B). The expression of TR-1 and QR-1 were analysed by Western blot analysis. The band density was quantified using the Quantity One®. Data are the average of 3 experiments (±SD).
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HHL-5 cells were treated with either SFN (5 μM), or CdSe (20 μM) and in their combination, i.e. pre-treatment SFN + CdSe for 24 h. DMSO (0.1%) as control. Total RNA was isolated using a GenElute™ total mammalian RNA kit (Sigma, UK). MT-1A mRNA was determined by TaqMan real-time PCR assays. The bar graphs represent means ± SD of three replicates. Statistical significance from the control, **p< 0.01.
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HHL-5 cells were pre-incubated with 3-MA (5 mM) for 6 h and then exposed to 5 μM SFN for 24 h. There was then a further 24 h exposure with 20 μM CdSe QDs. Cytotoxicity was measured by MTT assay. Data are shown as means ± SD (n = 6) (**P<0.01).
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The authors are extremely grateful to Dr Arvind Patel (Glasgow, UK) for providing the immortalized human hepatocytes, to Dr Noboru Mizushima (Tokyo, Japan) for providing MEF (atg5-/-), Japan. The authors also wish to thank Mr Jim Bacon, Dr Stuart Rushworth and Dr Andrew Goldson for helpful discussions during the preparation of this manuscript.