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The chemopreventive effects of aged garlic extract against cadmium-induced toxicity
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 2 ( 2 0 1 1 ) 266–274
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The chemopreventive effects of aged garlic extract against cadmium-induced toxicity Akeem O. Lawal ∗ , Elizabeth M. Ellis Strathclyde Institute of Pharmacy and Biomedical Sciences University of Strathclyde, 204 George Street Glasgow G1 1XW, United Kingdom
a r t i c l e
i n f o
a b s t r a c t
Article history:
Garlic has been reported in many previous studies as a potent chemopreventive agent. The
Received 12 January 2011
protective effect of garlic has been ascribed to the presence of organosulphur compounds
Received in revised form 7 May 2011
(OSC). In this study, the efficacy of aged garlic extract (AGE) compared to diallyl disulfide
Accepted 28 May 2011
(DADS) in protecting against toxicity induced by cadmium (Cd) in 1321N1 and HEK293 cells
Available online 13 June 2011
was investigated. The involvement of the transcription factor Nrf2 in this protection was also examined. The results show that AGE significantly prevented loss of cell viability in Cd-
Keywords:
treated 1321N1 and HEK293 cells. In comparison DADS had no significant effect in protecting
Nrf2
HEK293 cells but did protect 1321N1 cells. AGE significantly reduced Cd-induced TBARS
Cadmium
production and LDH leakage in the two cell lines, and AGE and DADS both increased GSH
DADS
levels in Cd-treated cell lines. Pre-treatment of cells with AGE or DADS increased expression
AGE
of the protective enzyme NAD(P)H:quinone oxidoreductase (NQO1), and this was associated
Reactive oxygen species
with the accumulation of the transcription factor Nrf2. These results show that AGE and
Chemoprevention
DADS have beneficial effects against Cd-induced toxicity, and this protection appears to
Cytoprotection
be mediated via induction of cytoprotective enzymes in an Nrf2-dependent manner. This indicates the potential for using AGE as a chemoprevention strategy for Cd toxicity. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Cadmium (Cd) is a heavy metal that is widely distributed in the environment. It is a serious industrial and environmental toxicant known to be carcinogenic to various organs in the body (Waalkes, 2000; Waisberg et al., 2003). The main industrial sources of Cd include refinery, smelting of metals such as copper and zinc, combustion of fossil fuel and nickel–cadmium battery manufacture and disposal.
Apart from industrial sources, cigarette smoke constitutes a major non-occupational source (WHO, 1992). Cd induces both necrotic and apoptotic cell death, dependent on dose and exposure time (Galan et al., 2001). Although it has multi-organ damaging effect, the risk and target organs depend mainly on the form of Cd and route of exposure. Chronic and acute Cd affects mainly the kidney, liver, cardiovascular and nervous system, and intestine (WHO, 1992). In previous work, we and others have established that at certain concentrations, Cd induces apoptotic or necrotic cell death in cell lines, and
Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; MDA, malondialdehyde; SDS-PAGE, sodium deodecyl sulphate polyacrylamide gel electrophoresis; OSC, organosulfur compounds; DADS, diallyl sulfide; AGE, aged garlic extract; SAC, S-allylcysteine; SAMC, S-allylmercaptocysteine; Cd, cadmium; GSH, glutathione; NQO1, NAD(P):quinone oxidoreductase. ∗ Corresponding author. Present address: UCLA Division of Cardiology, 10833 Le Conte Ave, A2-237 CHS, Los Angeles, CA 90095-1679, USA. Tel.: +1 310 866 6136/424 228 4032. E-mail address: [email protected] (A.O. Lawal). 1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.05.012
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that the mechanism of toxicity involves GSH depletion and oxidative stress (Lawal and Ellis, 2010). The use of chemopreventive agents has been exploited in the therapy against the toxic effect of many compounds including heavy metals. A range of plants, vegetables and synthetic compounds have been screened in order to ascertain their potential in the prevention and cure of many diseases and conditions (WCRF and AICR, 1997). One plant studied extensively is garlic (Allium sativum), which has been used since ancient times as a cure for many diseases (Block, 1985). Garlic is said to possess antibacterial, antifungal, antithrombotic, antihypertensive, anticarcinogenic and antioxidant properties (Wei et al., 1998). Epidemiological studies have consistently shown a relationship between intake of high quantity of garlic and a reduced incidence of cancer in most tissues especially in the stomach and colon (Fleischauer and Arab, 2001). The garlic plant contains both water- and lipid-soluble organosulfur compounds (OSC) which have been reported to be responsible for its therapeutic properties (Block, 1985). Preparation of an aqueous extract of garlic over a long period of time (10–20 months) yields aged garlic extract or AGE, which has enhanced antioxidant power (Moriguchi et al., 1997). AGE does not contain allicin, but does contain lipidsoluble allyl sulfides (Amagase, 1998; Imai et al., 1994), as well as a range of water-soluble OSC, including the allyl amino acid derivatives S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC). SAC and SAMC are generated during the garlic ageing process, meaning these potent antioxidant compounds are present at much higher concentrations in AGE than in fresh garlic. Garlic has been noted previously to protect against the toxic effects of Cd toxicity, both in animal models and in cell lines (Cha, 1987; Suru, 2008; Obioha et al., 2009). Several studies have identified diallyl sulfide and diallyl tetrasulfide as components of garlic extract that provide protection against Cd (Ponnusamy and Pari, in press; Sadik, 2008). Of particular interest has been the ability of garlic and diallyl sulfides to protect against cadmium-induced testicular damage (Ola-Mudathir et al., 2008), nephrotoxicity (Suru, 2008), and brain toxicity (Pari and Murugavel, 2007). Although protective effects have been observed, the mechanism by which garlic and its constituents protects against Cd toxicity at the molecular level is largely unknown. In addition, the protective effects of AGE in comparison to diallyl disulfides are not known. This present study therefore examines the effect of AGE in comparison to diallyl disulfide (DADS) in the protection against Cd toxicity in two cell lines, with the aim of investigating the mechanism of protection, and in particular to define the role of the transcription factor Nrf2 in mediating this process.
2.
Materials and methods
2.1.
Chemicals
Most chemicals were obtained from Sigma–Aldrich, UK. All the antibodies were obtained from Santa Cruz Biotechnology, UK. Horseradish peroxidase-conjugated goat anti-rabbit was obtained from Bio-Rad, UK. Acrylamide was obtained
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from Severn Biotech Ltd, UK. Nitrocellulose membranes were obtained from GE Healthcare.
2.2.
Preparation of garlic extracts
Garlic bulbs were purchased in Ondo State, Nigeria. Aged garlic extract (AGE) was prepared by soaking the crushed garlic in distilled water and allow the extraction to occur for 12 months at room temperature. The extracts were dried in a rotary evaporator at 25 ◦ C and used at concentration of 100 g/ml. Diallyl disulfide was prepared in 0.1% DMSO and used at a final concentration of 100 M.
2.3.
Cell culture
1321NI human astrocytoma cells (Clark et al., 1975) and HEK 293 human embryonic kidney cells (Graham et al., 1977) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). HEK 293 and 1321NI cell lines were all grown in Dulbecco’s modified Eagle’s medium (DMEM). 1321N1 and HEK 293 cells medium was supplemented with 10% FBS, 1% penicillin–streptomycin and 1% glutamine. The cells were maintained at 35 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. For cell viability measurements, cells were plated in 96-well plates at a concentration of 3.6 × 103 cells/well. For the preparation of cell extracts, cells were plated in 6-well plates at a density of 106 cells/well. Cells were allowed to attach for 24 h before treatment with either 100 g/ml AGE or 100 M of DADS for 24 h. After the incubation period, the cell monolayer was washed with PBS and 5, 10 and 50 M CdCl2 was added for a further 24 h as required.
2.4.
Cell viability assay
Cell viability was assessed by the MTT assay method (Mossmann, 1983). The cells were treated with compound for 24 h and/or Cd for 24 h in 96 well plates. 20 l of MTT (1.2 mg/ml) was added to each well and allowed to incubate for 4 h at 37 ◦ C. After incubation, the media were aspirated and 100 l of DMSO was added to each well to dissolve the formazan. Cells were then incubated for 10 min at 37 ◦ C and absorbance read at 560 nm with a microplate spectrophotometer. Results were expressed as mean value of percentage of untreated control.
2.5.
LDH leakage
Cytotoxicity was also assessed by the LDH leakage assay according to the method of Decker and Lohmann-Matthes (1988) by following the reduction of NAD by LDH using a tetrazolium dye with characteristic absorption at 490 nm. Cells were treated with compound for 24 h and/or Cd for 24 h in 96 well plates. The media was aspirated and centrifuged at 3000 × g for 5 min. LDH activity in the aliquot of the supernatant was determined using an LDH assay kit from Sigma. The reactions were carried out in 96 well plate containing 30 l of the supernatant and 60 l of LDH assay mixture and incubated at room temperature for 20–30 min. Absorbance was measured using a microplate spectrophotometer at 490 nm with background absorbance at 690 nm. The
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background absorbance was subtracted from the absorbance at 490 nm. Results were presented as mean value of percentage of control.
2.6.
Lipid peroxidation assay
A thiobarbituric acid reactive species (TBARS) assay was used to measure the extent of malondialdehyde (MDA) formed as a result of membrane lipid peroxidation. The assay was based on the formation of a pink coloured complex in a reaction between malondiadehyde formed during lipid peroxidation and thiobarbituric acid (TBA). Briefly, cells were seeded in 6 well plates at a density of 106 cells/well. Cell extracts were prepared according to the freeze-thaw method (Lyon et al., 2007). Briefly, 100 l cell extract was added to 1.4 ml TBA reagent (0.375% TBA, 0.3 M Sodium acetate and 0.15% SDS) and incubates for 60 min at 90 ◦ C. The mixture was allowed to cool on ice after the incubation and then centrifuge at 13,000 × g for 10 min. 1 ml of the supernatant was removed and absorbance read at 532 nm. The amount of MDA formed was extrapolated from a standard MDA curve and result was expressed as mol MDA/ml/mg protein.
2.7.
2.9.
Statistical analysis
Results were analysed using one way analysis of variants (ANOVA). Comparison between groups were carried out using Dunnet’s multiple comparison tests with significant difference at p < 0.005, p < 0.01, p < 0.05. Statistical analysis was carried out using GraphPad Prism software. Comparison within group was carried out using the unpaired student’s t test.
3.
Results
3.1.
Effect of AGE on cell viability after CdCl2 exposure
Glutathione (GSH) determination
GSH level was determined spectrophotometrically according to the method of Jollow et al. (1974). The method was based on the formation of a stable yellow coloured compound in a reaction between Ellman’s reagent (5,5 -dithiobis (-2-nitrobenzoic acid)) and GSH to give a coloured compound that has a characteristic absorption at 412 nm. Briefly, cells were seeded in 6 well plates at a density of 106 cells/well. After treatment, cell extracts were prepared according to the freeze-thaw method (Lyon et al., 2007). Equal volume of 4% sulfosalicylic acid was added to the cell extract in order to deproteinised the sample by removing other sulhydryl group containing proteins which can interfere with the reaction. The mixture was then centrifuge at 17,000 × g for 15 min at 2 ◦ C. 100 l of the supernatant was added to 900 l of Ellman’s reagent in a 1 ml cuvette and absorbance was measured at 412 nm. The amount of GSH in the sample was extrapolated from a standard GSH curve and result was expressed as g/mg cell protein.
2.8.
with Goat anti-Rabbit IG-horseradish peroxidase conjugate antibody (Bio-Rad) for 1 h at room temperature. The membrane was wash 3 times with 1× TBSTween and once with 10× TBS. The membrane was then developed using enhanced chemoluminescence (ECL) solutions (Lyon et al., 2007). Protein expression was quantified with an Image reader LAS 3000. Band intensity was quantified using the ImageJ software and protein expression was represented as relative to control.
Two cell lines were chosen to represent target tissues for Cd toxicity: 1321N1 human astrocytoma cells (Clark et al., 1975) and HEK 293 human embryonic kidney cells (Graham et al., 1977). As previously shown, exposure of both cell lines to Cd in the form of CdCl2 for 24 h led to significant loss of cell viability as determined by the MTT assay, and this was most acute at concentrations of 25–50 M CdCl2 (Fig. 1) (Lawal and Ellis, 2010). To determine the effect of AGE in protecting against Cdinduced toxicity, cells were pre-treated with either 100 g/ml AGE or 100 M DADS as a comparison for 24 h before exposure to Cd for 24 h. Cell viability was then determined using the MTT assay. The results in Fig. 1 show that pre-treatment of cells with AGE significantly protects HEK293 cells against Cd toxicity at all concentrations used. DADS pre-treatment did not lead to a significant increase in protection in HEK293 cells. AGE pre-treatment also protected 1321N1 cells, as did treatment with DADS (Fig. 1B). These set of data show that 100 g/ml AGE is more potent in protecting cells from Cd toxicity than 100 M DADS.
SDS-PAGE and Western blot analysis
Cells were plated in 6 well plates at a density of 106 cells/well followed by treatment as appropriate. Cells were then lysed in 200 l cell lysis buffer containing Tris Cl (pH 6.8) (3.13 ml), Sodium dodecyl sulphate (SDS) (2 g), glycerol (9 ml), mercaptoethanol (5 ml), bromophenol blue (0.1%) (1 ml) and distilled water (31.87 ml). The cells were then heated at 100 ◦ C for 5 min and centrifuge. Protein determination was carried out using a modified Bradford method (Bradford, 1976) and an equal amount of protein (10 g) was then separated by polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels (Laemmli, 1970). The separated proteins were then transferred to nitrocellulose membrane (Hybond ECL) for 2 h at 200 mA. After 1 h incubation with the specific antibodies, the membranes were washed 4 times with 1× TBSTween [10× TBS (100 ml), Tween 20 (2 ml) and distilled water] before incubation
3.2. Effects of AGE on lactate dehydrogenase (LDH) leakage after CdCl2 exposure In order to further assess the protective effect of AGE against Cd cytotoxicity, the LDH leakage assay was used as a measure of cell membrane damage. Rupture of the cell membrane occurs when cells are significantly damaged and indicates necrotic effects. The results in Fig. 2 show that as expected treatment of HEK293 cells with Cd led to significant increases in LDH leakage, particularly at 50 M CdCl2 which we have shown previously to cause necrosis. The presence of AGE and DADS resulted in a reduction in LDH leakage in HEK 293 cells when compared with cells exposed to 10 and 50 M CdCl2 (Fig. 2A). LDH leakage was also reduced in 1321N1 cells by treatment with AGE and DADS (Fig. 2B). The protection was highest in AGE pre-treated 1321N1 cells exposed to 50 M
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Fig. 1 – Effect of AGE and DADS on cell viability in the presence of CdCl2 . (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h before exposure to 5, 10, 25 and 50 M CdCl2 for 24 h. Cell viability was determined using the MTT assay as described in Section 2; data represent the mean value (n = 6 of individual experiments done in triplicate) of percentage of control ±SD. Asterisks indicate significant compared with untreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
CdCl2 and lowest in AGE pre-treated 1321N1 cells exposed to 10 M CdCl2 (Fig. 2B). These data show that pre-treatment of these two cell lines with AGE and DADS has beneficial effects against Cd-induced membrane damage especially at 10 and 50 M Cd.
3.3. Effects of AGE on lipid peroxidation after CdCl2 exposure In order to evaluate the protective effect of AGE on membrane lipid damage induced by Cd, malondialdehyde levels were determined by measuring thiorbarbituric acid reactive substance (TBARS). Exposure to Cd led to a significant increase in MDA in both cell lines, particular at concentrations of 10 and 50 M CdCl2 . Cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h prior to 24 h exposure to Cd. In HEK 293 cells, there was no significant reduction in MDA levels after exposure to 5 M CdCl2 in the absence and presence of AGE and DADS (Fig. 3A). However MDA levels were significantly
Fig. 2 – Effect of AGE and DADS on LDH leakage in the presence of CdCl2 . (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h before exposure to 5, 10, 25 and 50 M CdCl2 for 24 h. Cell cytotoxicity was determined by the LDH leakage as described in Section 2; data represent the mean value (n = 6 of individual experiments done in triplicate) of percentage of control ±SD. Asterisks indicate significant compared with untreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
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Fig. 3 – Effect of AGE and DADS on lipid peroxidation in the presence of CdCl2 . (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h before exposure to 5, 10, 25 and 50 M CdCl2 for 24 h. Lipid peroxidation was determined by the TBARS assay as described in Section 2; data represent the mean value (n = 6 of individual experiments done in triplicate) of percentage of control ±SD. Asterisks indicate significant compared with nonpretreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
Fig. 4 – Effect of AGE and DADS on GSH levels in the presence of CdCl2 . (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h before exposure to 50 M CdCl2 for 24 h. GSH levels were determined as described in Section 2; data represent the mean value (n = 6 of individual experiments done in triplicate) of percentage of control ±SD. Asterisks indicate significant compared with nonpretreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
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reduced in AGE and DADS pre-treated HEK 293 cells exposed to 10 and 50 M CdCl2 compared to the non-pretreated cells (Fig. 3A). The results also show that there was a significant reduction in malondialdehyde levels in AGE pre-treated 1321N1 cells when compared with the non-pretreated cells exposed to CdCl2 (Fig. 3B). The greatest effect was observed in AGE pre-treated 1321N1 cells exposed to 50 M CdCl2 . DADS pre-treatment did not appear to affect MDA levels in 1321N1 cells exposed to 50 M CdCl2 but could lower MDA levels at 10 M CdCl2 (Fig. 3B). These results suggest that both AGE and DADS are effective in protecting 1321N1 and HEK 293 cells from CdCl2 -induced lipid peroxidation. However in both cell lines, 100 g/ml AGE was more effective than 100 M DADS in protecting cells, particular at higher concentrations of Cd.
3.4.
Effects of AGE on GSH levels after CdCl2 exposure
GSH is an antioxidant that reduces reactive oxygen species and thereby serves as a first line of defence against free radical attack. Treatment of cells with 50 M CdCl2 led to a reduction in GSH levels in both cell lines as determined using Ellman’s reagent. In order to determine the effect of AGE pre-treatment on total GSH levels after Cd exposure, cells were pre-treated with either AGE or DADS for 24 h followed by 24 h exposure to CdCl2 . Pre-treatment of HEK293 cells with 100 g/ml AGE
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resulted in a significant increase in GSH levels when compared with cells treated with 50 M CdCl2 alone (Fig. 4A). The presence of DADS also restored GSH levels (Fig. 4A). In 1321N1 cells, the effect of Cd treatment in reducing GSH levels was much greater, and both AGE and DADS were able to increase GSH levels (Fig. 4B). However the data suggest that DADS is more effective in enhancing GSH levels in 1321N1 and that AGE is more effective in HEK 293 cells.
3.5. Effects of AGE on NQO1 expression in 1321N1 and HEK293 cells In order to assess whether the presence of AGE has any effect on the expression levels of the protective enzyme NAD(P)P:quinone oxidoreductase (NQO1), 1321N1 and HEK293 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h, and Western blot analysis was performed using an NQO1 specific antibody. In HEK293 cells, AGE pre-treatment significantly increased NQO1 expression by 2.1-fold when compared with control (Fig. 5), and DADS increased NQO1 expression by 3.2-fold. In 1321N1 cells, AGE pre-treatment led to a 2.6-fold significant increase in NQO1 expression, and DADS pre-treatment led to a 1.8-fold increase. These results indicate that AGE leads to the induction of protective enzymes that may account for some of the protection observed against Cd toxicity.
Fig. 5 – Effects of AGE and DADS on the expression of NQO1 in HEK293 and 1321N1 cells. (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h. cell extracts were prepared and 10 g of proteins were loaded on SDS-PAGE. Western blots were performed using NQO1 specific antibodies as described in Section 2. Protein loading was normalised with GAPDH antibody. The intensity of the bands was evaluated with ImageJ analysis software and relative protein expressions were compared to control. Data represent the mean value (n = 3 of individual experiments done in triplicate) of percentage of untreated control ±SD. Asterisks indicate significant compared with nonpretreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
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Fig. 6 – Effect of AGE and DADS on the accumulation of Nrf2 in HEK293 and 1321N1 cells. (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h. cell extracts were prepared and 10 g of proteins were loaded on SDS-PAGE. Western blots were performed using Nrf2 specific antibodies as described in Section 2. Protein loading was normalised with GAPDH antibody. The intensity of the bands was evaluated with ImageJ analysis software and relative protein expressions were compared to control. Data represent the mean value (n = 3 of individual experiments done in triplicate) of percentage of untreated control ±SD. Asterisks indicate significant compared with nonpretreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
3.6. Effects of AGE on Nrf2 expression in 1321N1 and HEK 293 cells To determine the mechanism by which NQO1 is induced, the levels of the transcription factor Nrf2 were investigated following AGE pretreatment. In HEK 293 cells, levels of Nrf2 increased significantly by 9.5-fold following AGE pretreatment, and by 1.5-fold following DADS pretreatment (Fig. 6). In 1321N1 cells, AGE pretreatment increased Nrf2 levels by 5.4-fold, and there was a 3.79-fold significant increase in Nrf2 levels in the presence of DADS in 1321N1 cells when compared with untreated control. In both cases, AGE pretreatment was significantly better at increasing Nrf2 levels. These sets of data suggest that AGE and DADS enhanced induction of Nrf2 and this induction leads to enhanced transcription of NQO1 which may be important in protecting cells from Cd.
4.
Discussion
In this present study the efficacy of AGE in protecting against Cd-induced toxicity in two cell lines was examined and compared to DADS, an organosulfur compound present in garlic.
The results show that AGE enhanced survival of 1321N1 and HEK 293 cells exposed to up to 50 M CdCl2 , and both AGE and DADS prevented Cd-induced cell damage. AGE and DADS were also found to inhibit MDA formation induced by Cd, indicating that these compounds were able to prevent the elevation of ROS and subsequent lipid peroxidation events. This is the first report to show that AGE can protect against Cd toxicity in these cell lines, and provides a powerful chemoprevention strategy for Cd exposure. The protective effects of AGE have been investigated previously in terms of preventing other types of cell damage, including oxidative stress, thermal stress and damage cause by other toxic compounds. For example, AGE and SAC have been shown to protect endothelial cells against damage caused by oxidized LDL-induced injury (Ide and Lau, 2001). Similar to the protection we observe against Cd, AGE and SAC prevented the loss of cell viability in endothelial cells, reduced LDH leakage, inhibited reactive oxygen species (ROS) production and prevented MDA formation (Ide and Lau, 2001; Ide and Lau, 1997). How might AGE protect against Cd toxicity? Cd is known to cause a range of biochemical effects in cells, including oxidative stress and depletion in GSH levels. GSH is the main
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thiol-containing antioxidant present in cells, and its depletion renders the cells vulnerable to oxidative damage. Therefore one possible mechanism of protection against Cd might be through enhancing GSH levels in cells. The presence of OSC in garlic extract can serve as an additional source of available electrons for the removal of electrophiles. Therefore, the net effect of the administration of OSC in garlic is to protect GSH pools. We have shown here that cells pre-treated with AGE showed a significant elevation in GSH levels. Other studies have shown similar effects. For example, in one study using rats as a model, aqueous garlic (GA) extract was shown to restore GSH levels during thermal injury (Sener et al., 2003). Similarly, AGE and SAC were found to prevent GSH depletion in endothelial cells exposed to oxidized LDL (Ide and Lau, 2001; Geng and Lau, 1997), and diallyl tetrasulfide was reported to protect rat brain against Cd toxicity by increasing GSH (Pari and Murugavel, 2007; Sheen et al., 1996). It is therefore likely that the OSC present in AGE are acting in a similar way in HEK293 and 1321N1 cells. This is the first study to show that AGE can prevent GSH depletion due to Cd treatment. By maintaining intracellular GSH levels, AGE can prevent the most damaging effects of oxidative stress, reduce MDA formation, and protect cells against necrosis at high concentrations of Cd. This explains the ability of AGE to prevent necrosis-associated LDH leakage. Many chemopreventive agents can also exert their action via the induction of detoxifying enzymes, leading to an increase in metabolism via reactions such as oxidation, reduction, hydrolysis and conjugation. These events can decrease the toxicity of reactive compounds and intermediates and facilitate their removal from the body (Kensler, 1997). In this study, we have shown that AGE and DADS pre-treatment leads to the induction of a protective response in 1321N1 and HEK293 cell lines, as exemplified by increased expression of NQO1. This is in agreement with previous studies that have shown that the diallyl sulfides upregulate NQO1 and heme oxygenase 1 expression in human hepatoma HepG2 cells (Chen et al., 2004). However, our work is the first to show that AGE is effective in inducing NQO1, and that the induction of this adaptive response is associated with protection against Cd toxicity. The induction of detoxifying enzymes has been reported to be mediated via the action of Nrf2, a member of the basic leucine zipper transcription factor family, that regulates transcription by binding to an antioxidant response element (ARE) present in promoter regions (Venugopal and Jaiswal, 1998). Under normal conditions, Nrf2 is not easily detectable in the cell because it is rapidly sequestered by a protein known as Keap1, which targets it for degradation (Itoh et al., 1999; Kobayashi et al., 2004). However, under inducing conditions Keap1 dissociates from Nrf2, allowing it to accumulate and activate transcription in the nucleus (Nguyen et al., 2009). We investigated whether Nrf2 is associated with the induction of NQO1 by AGE. Our results have shown that AGE can significantly increase Nrf2 levels in 1321N1 and HEK293 cells, similar to previous studies that have shown that diallyl sulfides increase Nrf2 levels in HepG2 cells (Chen et al., 2004; Lee and Surh, 2005). However, ours is the first study to show that AGE can stabilize Nrf2 in cell lines, and provides evidence
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that this mechanism is involved in the induction of protective enzymes, and leads to the increased survival of Cd exposed cells. In summary, the data obtained from this study show that AGE has beneficial actions against Cd-induced toxicity in 1321N1 and HEK 293 cells, and these include prevention of membrane damage and reduction in lipid peroxidation. These protective effects are likely to be mediated at least in part by the enhanced induction of the cytoprotective enzymes through the stabilization of Nrf2 in 1321N1 and HEK 293 cells.
Conflict of interest This work presents no conflict of interest.
Acknowledgement Akeem Lawal was funded by a Commonwealth Scholarship (UK).
references
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Imai, J., Ide, N., Nagae, S., Moriguchi, T., Matsuura, H., Itakura, Y., 1994. Antioxidant and radical scavenging effects of aged garlic extract and its constituents. Planta Med. 60, 417–420. Itoh, K., Wakabayashi, N., Katoh, Y., Ishii, T., Igarashi, K., Engel, J.D., Yamamoto, M., 1999. Keap1 represses nuclear activation of antioxidant responsive element by Nrf2 through binding to the amino terminal Neh2 domain. Gene Dev. 13, 76–86. Jollow, D.J., Mitchell, J.R., Zampaglione, N., Gillette, J.R., 1974. Bromobenzene-induced liver necrosis, protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 11, 151–169. Kensler, T.W., 1997. Chemoprevention by inducers of carcinogen detoxification enzymes. Environ. Health Perspect. 105, 965–970. Kobayashi, A., Kang, M.I., Okawa, H., Ohtsuji, M., Zenke, Y., Chiba, T., Igarashi, K., Yamamoto, M., 2004. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol. Cell. Biol. 24, 7130–7139. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lawal, A.O., Ellis, E., 2010. Differential sensitivity and responsiveness of three human cell lines HepG2, 1321N1 and HEK 293 to cadmium. J. Toxicol. Sci. 35, 465–478. Lee, J.S., Surh, Y.J., 2005. Nrf2 as a novel molecular target for chemoprevention. Cancer Lett. 224, 171–184. Lyon, R.C., Johnston, S.M., Watson, D.G., McGarvie, G., Ellis, E.M., 2007. Synthesis and catabolism of gamma-hydroxybutyrate in SH-SY5Y human neuroblastoma cells: role of the aldo-keto reductase AKR7A2. J. Biol. Chem. 282, 25986–25992. Mossmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Moriguchi, T., Salto, H., Nishiyama, N., 1997. Anti-aging effect of aged garlic extract in the inbred brain atrophy mouse model. Clin. Exp. Pharmacol. Physiol. 24, 235–242. Nguyen, T., Nioi, P., Pickett, C.B., 2009. The Nrf2-antioxidant response element signalling pathway and its activation by oxidative stress. J. Biol. Chem. 284 (20), 13291– 13295. Obioha, U.E., Suru, S.M., Ola-Mudathir, K.F., Faremi, T.Y., 2009. Hepatoprotective potentials of onion and garlic extracts on cadmium-induced oxidative damage in rats. Biol. Trace Elem. Res. 129, 143–156.
Ola-Mudathir, K.F., Suru, S.M., Fafunso, M.A., Obioha, U.E., Faremi, T.Y., 2008. Protective roles of onion and garlic extracts on cadmium-induced changes in sperm characteristics and testicular oxidative damage in rats. Food Chem. Toxicol. 46, 3604–3611. Pari, L., Murugavel, P., 2007. Diallyl tetrasulfide improves cadmium induced alterations of acetylcholinesterase. ATPases and oxidative stress in brain of rats. Toxicology 234, 44–50. Ponnusamy, M., Pari L. Protective role of diallyl tetrasulfide on cadmium-induced testicular damage in adult rats: a biochemical and histological study. Toxicol. Ind. Health, in press. Sadik, N.A., 2008. Effects of diallyl sulfide and zinc on testicular steroidogenesis in cadmium-treated male rats. J. Biochem. Mol. Toxicol. 22, 345–353. Sener, G., Satyroglu, H., Sehirli, A.O., Kacmaz, A., 2003. Protective effect of aqueous garlic extract against oxidative organ damage in a rat model of thermal injury. Life Sci. 73, 81–91. Sheen, L.Y., Lii, C.K., Sheu, S.F., Meng, R.H., Tsai, S.J., 1996. Effect of the active principle of garlic-dially sulphide-on cell viability, detoxification capability and the antioxidant system of primary rat hepatocytes. Food Chem. Toxicol. 34, 971–978. Suru, S.M., 2008. Onion and garlic extracts lessen cadmium-induced nephrotoxicity in rats. Biometals 21, 623–633. Venugopal, R., Jaiswal, A.K., 1998. Nrf2 and Nrf1 in association with Jun proteins regulate antioxidant response element-mediated expression and coordinated induction of genes encoding detoxifying enzymes. Oncogene 17, 3145–3156. Waalkes, M.P., 2000. Cadmium carcinogenesis in review. J. Inorg. Biochem. 79, 241–244. Waisberg, M., Joseph, P., Hale, B., Beyersmann, D., 2003. Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192, 95–117. WCRF, AICR, 1997. Vegetables and fruits. In: World Cancer Research Fund, American Institute for Cancer Research (Eds.), Food, Nutrition and the Prevention of Cancer: A Global Perspective. American Institute for Cancer Research, Washington, DC, pp. 436–446. Wei, Z., Lau, B.H., Lau, S., 1998. Garlic inhibits free radical generation and augments antioxidant enzyme activity in vascular endothelial cells. Nutr. Res. 18, 61–70. WHO, 1992. Cadmium. Environmental Health Criteria, vol. 134. World Health Organisation.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/etap
The chemopreventive effects of aged garlic extract against cadmium-induced toxicity Akeem O. Lawal ∗ , Elizabeth M. Ellis Strathclyde Institute of Pharmacy and Biomedical Sciences University of Strathclyde, 204 George Street Glasgow G1 1XW, United Kingdom
a r t i c l e
i n f o
a b s t r a c t
Article history:
Garlic has been reported in many previous studies as a potent chemopreventive agent. The
Received 12 January 2011
protective effect of garlic has been ascribed to the presence of organosulphur compounds
Received in revised form 7 May 2011
(OSC). In this study, the efficacy of aged garlic extract (AGE) compared to diallyl disulfide
Accepted 28 May 2011
(DADS) in protecting against toxicity induced by cadmium (Cd) in 1321N1 and HEK293 cells
Available online 13 June 2011
was investigated. The involvement of the transcription factor Nrf2 in this protection was also examined. The results show that AGE significantly prevented loss of cell viability in Cd-
Keywords:
treated 1321N1 and HEK293 cells. In comparison DADS had no significant effect in protecting
Nrf2
HEK293 cells but did protect 1321N1 cells. AGE significantly reduced Cd-induced TBARS
Cadmium
production and LDH leakage in the two cell lines, and AGE and DADS both increased GSH
DADS
levels in Cd-treated cell lines. Pre-treatment of cells with AGE or DADS increased expression
AGE
of the protective enzyme NAD(P)H:quinone oxidoreductase (NQO1), and this was associated
Reactive oxygen species
with the accumulation of the transcription factor Nrf2. These results show that AGE and
Chemoprevention
DADS have beneficial effects against Cd-induced toxicity, and this protection appears to
Cytoprotection
be mediated via induction of cytoprotective enzymes in an Nrf2-dependent manner. This indicates the potential for using AGE as a chemoprevention strategy for Cd toxicity. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Cadmium (Cd) is a heavy metal that is widely distributed in the environment. It is a serious industrial and environmental toxicant known to be carcinogenic to various organs in the body (Waalkes, 2000; Waisberg et al., 2003). The main industrial sources of Cd include refinery, smelting of metals such as copper and zinc, combustion of fossil fuel and nickel–cadmium battery manufacture and disposal.
Apart from industrial sources, cigarette smoke constitutes a major non-occupational source (WHO, 1992). Cd induces both necrotic and apoptotic cell death, dependent on dose and exposure time (Galan et al., 2001). Although it has multi-organ damaging effect, the risk and target organs depend mainly on the form of Cd and route of exposure. Chronic and acute Cd affects mainly the kidney, liver, cardiovascular and nervous system, and intestine (WHO, 1992). In previous work, we and others have established that at certain concentrations, Cd induces apoptotic or necrotic cell death in cell lines, and
Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH, lactate dehydrogenase; MDA, malondialdehyde; SDS-PAGE, sodium deodecyl sulphate polyacrylamide gel electrophoresis; OSC, organosulfur compounds; DADS, diallyl sulfide; AGE, aged garlic extract; SAC, S-allylcysteine; SAMC, S-allylmercaptocysteine; Cd, cadmium; GSH, glutathione; NQO1, NAD(P):quinone oxidoreductase. ∗ Corresponding author. Present address: UCLA Division of Cardiology, 10833 Le Conte Ave, A2-237 CHS, Los Angeles, CA 90095-1679, USA. Tel.: +1 310 866 6136/424 228 4032. E-mail address: [email protected] (A.O. Lawal). 1382-6689/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2011.05.012
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that the mechanism of toxicity involves GSH depletion and oxidative stress (Lawal and Ellis, 2010). The use of chemopreventive agents has been exploited in the therapy against the toxic effect of many compounds including heavy metals. A range of plants, vegetables and synthetic compounds have been screened in order to ascertain their potential in the prevention and cure of many diseases and conditions (WCRF and AICR, 1997). One plant studied extensively is garlic (Allium sativum), which has been used since ancient times as a cure for many diseases (Block, 1985). Garlic is said to possess antibacterial, antifungal, antithrombotic, antihypertensive, anticarcinogenic and antioxidant properties (Wei et al., 1998). Epidemiological studies have consistently shown a relationship between intake of high quantity of garlic and a reduced incidence of cancer in most tissues especially in the stomach and colon (Fleischauer and Arab, 2001). The garlic plant contains both water- and lipid-soluble organosulfur compounds (OSC) which have been reported to be responsible for its therapeutic properties (Block, 1985). Preparation of an aqueous extract of garlic over a long period of time (10–20 months) yields aged garlic extract or AGE, which has enhanced antioxidant power (Moriguchi et al., 1997). AGE does not contain allicin, but does contain lipidsoluble allyl sulfides (Amagase, 1998; Imai et al., 1994), as well as a range of water-soluble OSC, including the allyl amino acid derivatives S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC). SAC and SAMC are generated during the garlic ageing process, meaning these potent antioxidant compounds are present at much higher concentrations in AGE than in fresh garlic. Garlic has been noted previously to protect against the toxic effects of Cd toxicity, both in animal models and in cell lines (Cha, 1987; Suru, 2008; Obioha et al., 2009). Several studies have identified diallyl sulfide and diallyl tetrasulfide as components of garlic extract that provide protection against Cd (Ponnusamy and Pari, in press; Sadik, 2008). Of particular interest has been the ability of garlic and diallyl sulfides to protect against cadmium-induced testicular damage (Ola-Mudathir et al., 2008), nephrotoxicity (Suru, 2008), and brain toxicity (Pari and Murugavel, 2007). Although protective effects have been observed, the mechanism by which garlic and its constituents protects against Cd toxicity at the molecular level is largely unknown. In addition, the protective effects of AGE in comparison to diallyl disulfides are not known. This present study therefore examines the effect of AGE in comparison to diallyl disulfide (DADS) in the protection against Cd toxicity in two cell lines, with the aim of investigating the mechanism of protection, and in particular to define the role of the transcription factor Nrf2 in mediating this process.
2.
Materials and methods
2.1.
Chemicals
Most chemicals were obtained from Sigma–Aldrich, UK. All the antibodies were obtained from Santa Cruz Biotechnology, UK. Horseradish peroxidase-conjugated goat anti-rabbit was obtained from Bio-Rad, UK. Acrylamide was obtained
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from Severn Biotech Ltd, UK. Nitrocellulose membranes were obtained from GE Healthcare.
2.2.
Preparation of garlic extracts
Garlic bulbs were purchased in Ondo State, Nigeria. Aged garlic extract (AGE) was prepared by soaking the crushed garlic in distilled water and allow the extraction to occur for 12 months at room temperature. The extracts were dried in a rotary evaporator at 25 ◦ C and used at concentration of 100 g/ml. Diallyl disulfide was prepared in 0.1% DMSO and used at a final concentration of 100 M.
2.3.
Cell culture
1321NI human astrocytoma cells (Clark et al., 1975) and HEK 293 human embryonic kidney cells (Graham et al., 1977) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). HEK 293 and 1321NI cell lines were all grown in Dulbecco’s modified Eagle’s medium (DMEM). 1321N1 and HEK 293 cells medium was supplemented with 10% FBS, 1% penicillin–streptomycin and 1% glutamine. The cells were maintained at 35 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. For cell viability measurements, cells were plated in 96-well plates at a concentration of 3.6 × 103 cells/well. For the preparation of cell extracts, cells were plated in 6-well plates at a density of 106 cells/well. Cells were allowed to attach for 24 h before treatment with either 100 g/ml AGE or 100 M of DADS for 24 h. After the incubation period, the cell monolayer was washed with PBS and 5, 10 and 50 M CdCl2 was added for a further 24 h as required.
2.4.
Cell viability assay
Cell viability was assessed by the MTT assay method (Mossmann, 1983). The cells were treated with compound for 24 h and/or Cd for 24 h in 96 well plates. 20 l of MTT (1.2 mg/ml) was added to each well and allowed to incubate for 4 h at 37 ◦ C. After incubation, the media were aspirated and 100 l of DMSO was added to each well to dissolve the formazan. Cells were then incubated for 10 min at 37 ◦ C and absorbance read at 560 nm with a microplate spectrophotometer. Results were expressed as mean value of percentage of untreated control.
2.5.
LDH leakage
Cytotoxicity was also assessed by the LDH leakage assay according to the method of Decker and Lohmann-Matthes (1988) by following the reduction of NAD by LDH using a tetrazolium dye with characteristic absorption at 490 nm. Cells were treated with compound for 24 h and/or Cd for 24 h in 96 well plates. The media was aspirated and centrifuged at 3000 × g for 5 min. LDH activity in the aliquot of the supernatant was determined using an LDH assay kit from Sigma. The reactions were carried out in 96 well plate containing 30 l of the supernatant and 60 l of LDH assay mixture and incubated at room temperature for 20–30 min. Absorbance was measured using a microplate spectrophotometer at 490 nm with background absorbance at 690 nm. The
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background absorbance was subtracted from the absorbance at 490 nm. Results were presented as mean value of percentage of control.
2.6.
Lipid peroxidation assay
A thiobarbituric acid reactive species (TBARS) assay was used to measure the extent of malondialdehyde (MDA) formed as a result of membrane lipid peroxidation. The assay was based on the formation of a pink coloured complex in a reaction between malondiadehyde formed during lipid peroxidation and thiobarbituric acid (TBA). Briefly, cells were seeded in 6 well plates at a density of 106 cells/well. Cell extracts were prepared according to the freeze-thaw method (Lyon et al., 2007). Briefly, 100 l cell extract was added to 1.4 ml TBA reagent (0.375% TBA, 0.3 M Sodium acetate and 0.15% SDS) and incubates for 60 min at 90 ◦ C. The mixture was allowed to cool on ice after the incubation and then centrifuge at 13,000 × g for 10 min. 1 ml of the supernatant was removed and absorbance read at 532 nm. The amount of MDA formed was extrapolated from a standard MDA curve and result was expressed as mol MDA/ml/mg protein.
2.7.
2.9.
Statistical analysis
Results were analysed using one way analysis of variants (ANOVA). Comparison between groups were carried out using Dunnet’s multiple comparison tests with significant difference at p < 0.005, p < 0.01, p < 0.05. Statistical analysis was carried out using GraphPad Prism software. Comparison within group was carried out using the unpaired student’s t test.
3.
Results
3.1.
Effect of AGE on cell viability after CdCl2 exposure
Glutathione (GSH) determination
GSH level was determined spectrophotometrically according to the method of Jollow et al. (1974). The method was based on the formation of a stable yellow coloured compound in a reaction between Ellman’s reagent (5,5 -dithiobis (-2-nitrobenzoic acid)) and GSH to give a coloured compound that has a characteristic absorption at 412 nm. Briefly, cells were seeded in 6 well plates at a density of 106 cells/well. After treatment, cell extracts were prepared according to the freeze-thaw method (Lyon et al., 2007). Equal volume of 4% sulfosalicylic acid was added to the cell extract in order to deproteinised the sample by removing other sulhydryl group containing proteins which can interfere with the reaction. The mixture was then centrifuge at 17,000 × g for 15 min at 2 ◦ C. 100 l of the supernatant was added to 900 l of Ellman’s reagent in a 1 ml cuvette and absorbance was measured at 412 nm. The amount of GSH in the sample was extrapolated from a standard GSH curve and result was expressed as g/mg cell protein.
2.8.
with Goat anti-Rabbit IG-horseradish peroxidase conjugate antibody (Bio-Rad) for 1 h at room temperature. The membrane was wash 3 times with 1× TBSTween and once with 10× TBS. The membrane was then developed using enhanced chemoluminescence (ECL) solutions (Lyon et al., 2007). Protein expression was quantified with an Image reader LAS 3000. Band intensity was quantified using the ImageJ software and protein expression was represented as relative to control.
Two cell lines were chosen to represent target tissues for Cd toxicity: 1321N1 human astrocytoma cells (Clark et al., 1975) and HEK 293 human embryonic kidney cells (Graham et al., 1977). As previously shown, exposure of both cell lines to Cd in the form of CdCl2 for 24 h led to significant loss of cell viability as determined by the MTT assay, and this was most acute at concentrations of 25–50 M CdCl2 (Fig. 1) (Lawal and Ellis, 2010). To determine the effect of AGE in protecting against Cdinduced toxicity, cells were pre-treated with either 100 g/ml AGE or 100 M DADS as a comparison for 24 h before exposure to Cd for 24 h. Cell viability was then determined using the MTT assay. The results in Fig. 1 show that pre-treatment of cells with AGE significantly protects HEK293 cells against Cd toxicity at all concentrations used. DADS pre-treatment did not lead to a significant increase in protection in HEK293 cells. AGE pre-treatment also protected 1321N1 cells, as did treatment with DADS (Fig. 1B). These set of data show that 100 g/ml AGE is more potent in protecting cells from Cd toxicity than 100 M DADS.
SDS-PAGE and Western blot analysis
Cells were plated in 6 well plates at a density of 106 cells/well followed by treatment as appropriate. Cells were then lysed in 200 l cell lysis buffer containing Tris Cl (pH 6.8) (3.13 ml), Sodium dodecyl sulphate (SDS) (2 g), glycerol (9 ml), mercaptoethanol (5 ml), bromophenol blue (0.1%) (1 ml) and distilled water (31.87 ml). The cells were then heated at 100 ◦ C for 5 min and centrifuge. Protein determination was carried out using a modified Bradford method (Bradford, 1976) and an equal amount of protein (10 g) was then separated by polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels (Laemmli, 1970). The separated proteins were then transferred to nitrocellulose membrane (Hybond ECL) for 2 h at 200 mA. After 1 h incubation with the specific antibodies, the membranes were washed 4 times with 1× TBSTween [10× TBS (100 ml), Tween 20 (2 ml) and distilled water] before incubation
3.2. Effects of AGE on lactate dehydrogenase (LDH) leakage after CdCl2 exposure In order to further assess the protective effect of AGE against Cd cytotoxicity, the LDH leakage assay was used as a measure of cell membrane damage. Rupture of the cell membrane occurs when cells are significantly damaged and indicates necrotic effects. The results in Fig. 2 show that as expected treatment of HEK293 cells with Cd led to significant increases in LDH leakage, particularly at 50 M CdCl2 which we have shown previously to cause necrosis. The presence of AGE and DADS resulted in a reduction in LDH leakage in HEK 293 cells when compared with cells exposed to 10 and 50 M CdCl2 (Fig. 2A). LDH leakage was also reduced in 1321N1 cells by treatment with AGE and DADS (Fig. 2B). The protection was highest in AGE pre-treated 1321N1 cells exposed to 50 M
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Fig. 1 – Effect of AGE and DADS on cell viability in the presence of CdCl2 . (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h before exposure to 5, 10, 25 and 50 M CdCl2 for 24 h. Cell viability was determined using the MTT assay as described in Section 2; data represent the mean value (n = 6 of individual experiments done in triplicate) of percentage of control ±SD. Asterisks indicate significant compared with untreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
CdCl2 and lowest in AGE pre-treated 1321N1 cells exposed to 10 M CdCl2 (Fig. 2B). These data show that pre-treatment of these two cell lines with AGE and DADS has beneficial effects against Cd-induced membrane damage especially at 10 and 50 M Cd.
3.3. Effects of AGE on lipid peroxidation after CdCl2 exposure In order to evaluate the protective effect of AGE on membrane lipid damage induced by Cd, malondialdehyde levels were determined by measuring thiorbarbituric acid reactive substance (TBARS). Exposure to Cd led to a significant increase in MDA in both cell lines, particular at concentrations of 10 and 50 M CdCl2 . Cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h prior to 24 h exposure to Cd. In HEK 293 cells, there was no significant reduction in MDA levels after exposure to 5 M CdCl2 in the absence and presence of AGE and DADS (Fig. 3A). However MDA levels were significantly
Fig. 2 – Effect of AGE and DADS on LDH leakage in the presence of CdCl2 . (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h before exposure to 5, 10, 25 and 50 M CdCl2 for 24 h. Cell cytotoxicity was determined by the LDH leakage as described in Section 2; data represent the mean value (n = 6 of individual experiments done in triplicate) of percentage of control ±SD. Asterisks indicate significant compared with untreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
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Fig. 3 – Effect of AGE and DADS on lipid peroxidation in the presence of CdCl2 . (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h before exposure to 5, 10, 25 and 50 M CdCl2 for 24 h. Lipid peroxidation was determined by the TBARS assay as described in Section 2; data represent the mean value (n = 6 of individual experiments done in triplicate) of percentage of control ±SD. Asterisks indicate significant compared with nonpretreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
Fig. 4 – Effect of AGE and DADS on GSH levels in the presence of CdCl2 . (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h before exposure to 50 M CdCl2 for 24 h. GSH levels were determined as described in Section 2; data represent the mean value (n = 6 of individual experiments done in triplicate) of percentage of control ±SD. Asterisks indicate significant compared with nonpretreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
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reduced in AGE and DADS pre-treated HEK 293 cells exposed to 10 and 50 M CdCl2 compared to the non-pretreated cells (Fig. 3A). The results also show that there was a significant reduction in malondialdehyde levels in AGE pre-treated 1321N1 cells when compared with the non-pretreated cells exposed to CdCl2 (Fig. 3B). The greatest effect was observed in AGE pre-treated 1321N1 cells exposed to 50 M CdCl2 . DADS pre-treatment did not appear to affect MDA levels in 1321N1 cells exposed to 50 M CdCl2 but could lower MDA levels at 10 M CdCl2 (Fig. 3B). These results suggest that both AGE and DADS are effective in protecting 1321N1 and HEK 293 cells from CdCl2 -induced lipid peroxidation. However in both cell lines, 100 g/ml AGE was more effective than 100 M DADS in protecting cells, particular at higher concentrations of Cd.
3.4.
Effects of AGE on GSH levels after CdCl2 exposure
GSH is an antioxidant that reduces reactive oxygen species and thereby serves as a first line of defence against free radical attack. Treatment of cells with 50 M CdCl2 led to a reduction in GSH levels in both cell lines as determined using Ellman’s reagent. In order to determine the effect of AGE pre-treatment on total GSH levels after Cd exposure, cells were pre-treated with either AGE or DADS for 24 h followed by 24 h exposure to CdCl2 . Pre-treatment of HEK293 cells with 100 g/ml AGE
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resulted in a significant increase in GSH levels when compared with cells treated with 50 M CdCl2 alone (Fig. 4A). The presence of DADS also restored GSH levels (Fig. 4A). In 1321N1 cells, the effect of Cd treatment in reducing GSH levels was much greater, and both AGE and DADS were able to increase GSH levels (Fig. 4B). However the data suggest that DADS is more effective in enhancing GSH levels in 1321N1 and that AGE is more effective in HEK 293 cells.
3.5. Effects of AGE on NQO1 expression in 1321N1 and HEK293 cells In order to assess whether the presence of AGE has any effect on the expression levels of the protective enzyme NAD(P)P:quinone oxidoreductase (NQO1), 1321N1 and HEK293 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h, and Western blot analysis was performed using an NQO1 specific antibody. In HEK293 cells, AGE pre-treatment significantly increased NQO1 expression by 2.1-fold when compared with control (Fig. 5), and DADS increased NQO1 expression by 3.2-fold. In 1321N1 cells, AGE pre-treatment led to a 2.6-fold significant increase in NQO1 expression, and DADS pre-treatment led to a 1.8-fold increase. These results indicate that AGE leads to the induction of protective enzymes that may account for some of the protection observed against Cd toxicity.
Fig. 5 – Effects of AGE and DADS on the expression of NQO1 in HEK293 and 1321N1 cells. (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h. cell extracts were prepared and 10 g of proteins were loaded on SDS-PAGE. Western blots were performed using NQO1 specific antibodies as described in Section 2. Protein loading was normalised with GAPDH antibody. The intensity of the bands was evaluated with ImageJ analysis software and relative protein expressions were compared to control. Data represent the mean value (n = 3 of individual experiments done in triplicate) of percentage of untreated control ±SD. Asterisks indicate significant compared with nonpretreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
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Fig. 6 – Effect of AGE and DADS on the accumulation of Nrf2 in HEK293 and 1321N1 cells. (A) HEK 293 and (B) 1321N1 cells were pre-treated with either 100 g/ml AGE or 100 M DADS for 24 h. cell extracts were prepared and 10 g of proteins were loaded on SDS-PAGE. Western blots were performed using Nrf2 specific antibodies as described in Section 2. Protein loading was normalised with GAPDH antibody. The intensity of the bands was evaluated with ImageJ analysis software and relative protein expressions were compared to control. Data represent the mean value (n = 3 of individual experiments done in triplicate) of percentage of untreated control ±SD. Asterisks indicate significant compared with nonpretreated control (**p < 0.01) using one-way ANOVA with Dunnett’s post test.
3.6. Effects of AGE on Nrf2 expression in 1321N1 and HEK 293 cells To determine the mechanism by which NQO1 is induced, the levels of the transcription factor Nrf2 were investigated following AGE pretreatment. In HEK 293 cells, levels of Nrf2 increased significantly by 9.5-fold following AGE pretreatment, and by 1.5-fold following DADS pretreatment (Fig. 6). In 1321N1 cells, AGE pretreatment increased Nrf2 levels by 5.4-fold, and there was a 3.79-fold significant increase in Nrf2 levels in the presence of DADS in 1321N1 cells when compared with untreated control. In both cases, AGE pretreatment was significantly better at increasing Nrf2 levels. These sets of data suggest that AGE and DADS enhanced induction of Nrf2 and this induction leads to enhanced transcription of NQO1 which may be important in protecting cells from Cd.
4.
Discussion
In this present study the efficacy of AGE in protecting against Cd-induced toxicity in two cell lines was examined and compared to DADS, an organosulfur compound present in garlic.
The results show that AGE enhanced survival of 1321N1 and HEK 293 cells exposed to up to 50 M CdCl2 , and both AGE and DADS prevented Cd-induced cell damage. AGE and DADS were also found to inhibit MDA formation induced by Cd, indicating that these compounds were able to prevent the elevation of ROS and subsequent lipid peroxidation events. This is the first report to show that AGE can protect against Cd toxicity in these cell lines, and provides a powerful chemoprevention strategy for Cd exposure. The protective effects of AGE have been investigated previously in terms of preventing other types of cell damage, including oxidative stress, thermal stress and damage cause by other toxic compounds. For example, AGE and SAC have been shown to protect endothelial cells against damage caused by oxidized LDL-induced injury (Ide and Lau, 2001). Similar to the protection we observe against Cd, AGE and SAC prevented the loss of cell viability in endothelial cells, reduced LDH leakage, inhibited reactive oxygen species (ROS) production and prevented MDA formation (Ide and Lau, 2001; Ide and Lau, 1997). How might AGE protect against Cd toxicity? Cd is known to cause a range of biochemical effects in cells, including oxidative stress and depletion in GSH levels. GSH is the main
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thiol-containing antioxidant present in cells, and its depletion renders the cells vulnerable to oxidative damage. Therefore one possible mechanism of protection against Cd might be through enhancing GSH levels in cells. The presence of OSC in garlic extract can serve as an additional source of available electrons for the removal of electrophiles. Therefore, the net effect of the administration of OSC in garlic is to protect GSH pools. We have shown here that cells pre-treated with AGE showed a significant elevation in GSH levels. Other studies have shown similar effects. For example, in one study using rats as a model, aqueous garlic (GA) extract was shown to restore GSH levels during thermal injury (Sener et al., 2003). Similarly, AGE and SAC were found to prevent GSH depletion in endothelial cells exposed to oxidized LDL (Ide and Lau, 2001; Geng and Lau, 1997), and diallyl tetrasulfide was reported to protect rat brain against Cd toxicity by increasing GSH (Pari and Murugavel, 2007; Sheen et al., 1996). It is therefore likely that the OSC present in AGE are acting in a similar way in HEK293 and 1321N1 cells. This is the first study to show that AGE can prevent GSH depletion due to Cd treatment. By maintaining intracellular GSH levels, AGE can prevent the most damaging effects of oxidative stress, reduce MDA formation, and protect cells against necrosis at high concentrations of Cd. This explains the ability of AGE to prevent necrosis-associated LDH leakage. Many chemopreventive agents can also exert their action via the induction of detoxifying enzymes, leading to an increase in metabolism via reactions such as oxidation, reduction, hydrolysis and conjugation. These events can decrease the toxicity of reactive compounds and intermediates and facilitate their removal from the body (Kensler, 1997). In this study, we have shown that AGE and DADS pre-treatment leads to the induction of a protective response in 1321N1 and HEK293 cell lines, as exemplified by increased expression of NQO1. This is in agreement with previous studies that have shown that the diallyl sulfides upregulate NQO1 and heme oxygenase 1 expression in human hepatoma HepG2 cells (Chen et al., 2004). However, our work is the first to show that AGE is effective in inducing NQO1, and that the induction of this adaptive response is associated with protection against Cd toxicity. The induction of detoxifying enzymes has been reported to be mediated via the action of Nrf2, a member of the basic leucine zipper transcription factor family, that regulates transcription by binding to an antioxidant response element (ARE) present in promoter regions (Venugopal and Jaiswal, 1998). Under normal conditions, Nrf2 is not easily detectable in the cell because it is rapidly sequestered by a protein known as Keap1, which targets it for degradation (Itoh et al., 1999; Kobayashi et al., 2004). However, under inducing conditions Keap1 dissociates from Nrf2, allowing it to accumulate and activate transcription in the nucleus (Nguyen et al., 2009). We investigated whether Nrf2 is associated with the induction of NQO1 by AGE. Our results have shown that AGE can significantly increase Nrf2 levels in 1321N1 and HEK293 cells, similar to previous studies that have shown that diallyl sulfides increase Nrf2 levels in HepG2 cells (Chen et al., 2004; Lee and Surh, 2005). However, ours is the first study to show that AGE can stabilize Nrf2 in cell lines, and provides evidence
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that this mechanism is involved in the induction of protective enzymes, and leads to the increased survival of Cd exposed cells. In summary, the data obtained from this study show that AGE has beneficial actions against Cd-induced toxicity in 1321N1 and HEK 293 cells, and these include prevention of membrane damage and reduction in lipid peroxidation. These protective effects are likely to be mediated at least in part by the enhanced induction of the cytoprotective enzymes through the stabilization of Nrf2 in 1321N1 and HEK 293 cells.
Conflict of interest This work presents no conflict of interest.
Acknowledgement Akeem Lawal was funded by a Commonwealth Scholarship (UK).
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