Liposomal silymarin anti-oxidative and anti-apoptotic features in lung cells: An implication in cadmium toxicity

Heavy metals are serious toxicants, and environmental pollution increase the risk of their exposure to human. This study compared the role of silymarin nanoliposomes (SL-L) against cadmium (Cd) toxicity in normal MRC-5 and A 549 cancer cells. For this, MRC-5 and A 549 cells exposed to Cd at 25 and 0.25 µM respectively, were treated with various non-toxic SL-L concentrations (2.5, 5, 10 µM) and the cells viability, reactive oxygen species (ROS) generation, apoptosis and the levels of cleaved PARP and caspase-3 proteins were determined following incubation. Results indicated that Cd exposure signi�cantly increased apoptosis due to ROS generation, and showed greater toxicity on cancer cells compared to the normal cells. While SL-L at higher concentrations (25 µM and higher) exhibits pro-apoptotic features, lower concentrations (10 and 2.5 µM for MRC-5 and A 549 cancer cells, respectively) played a protective and anti-oxidant role in Cd induced toxicity in both cells. Further, lower SL-L was required to protect cancer cells against Cd toxicity. In general, treatment with SL-L signi�cantly improved the cell survival by decreasing ROS levels, cleaved PARP and caspase-3 in both MRC-5 and A 549 cells compared to free silymarin. Results demonstrated that SL-L potential in protecting against Cd-induced toxicity depends on concentration-dependent antioxidant and anti-apoptotic balance.


Introduction
Cadmium (Cd) is a divalent heavy metal that greatly contributes to the environmental contamination due to its non-biodegradable nature.So, it is considered as a serious health problem for both animals and human [1,2].Its occurrence in the environment is from agricultural and industrial sources coming from human activities including the use of fossil fuels, waste burning, mining performance, producing polyvinyl chloride plastic as well as nickel-Cd batteries [3].Two major sources of Cd compounds exposure are ingestion of the contaminated food and water including farm products and seafood, and cigarette smoking and inhalation.It has been shown that Cd could accumulate in plants and animal organs including lungs, liver and kidney with a very long biological half-life of about 25-30 years [4].In human, even a very low concentration of Cd can contribute to a well-de ned spectrum of diseases.

Various clinical manifestations of Cd intoxication reported in the past century include lungs damage in
Cd-exposed workers in 1930s to bone and kidney toxicity and the occurrence of Itai-itai disease due to Cd contaminated rice elds [5].Inhalation is the main causes of Cd exposure in humans and the inhaled Cd absorbed by lung tissue could lead to various lung injury including pulmonary damage, emphysema and lung cancer [4].
Cd is a known carcinogen and prooxidant compound affecting the cell proliferation, differentiation and apoptosis.Epidemiological data suggested that Cd-induced genotoxicity may be an important attribute to various types of cancers, including breast, lung, prostate, nasopharynx, pancreas, and kidney cancers.Further, Cd provoke Reactive Oxygen Species (ROS) generation, reduced glutathione (GSH) depletion, lipid degradation, protein denaturation and DNA damage [6][7][8].
The excretion of Cd and heavy metals largely depends on the presence of antioxidative agents and thiols that assist with Cd metallothionein-binding [9].Various preventive and therapeutic approaches have been reported to reduce Cd poisoning including natural decontamination of water or phytomediation detoxi cation of pollutants [10].Combination therapy using various chelating agents is also regarded as a promising therapeutic approach [11,12].Phytochelating is another central mechanism in metal detoxi cation by sequestering toxic agents.So, numerous antioxidants including NAC, zinc, methionine, and cysteine were used in conjunction with standard chelating agents to improve the excretion of Cd [9].Silymarin (SL), a polyphenol from milk thistle (Silybum marianum) plant is a combination of six lignans including silychristin (SC), silydianin(SD), silybin A(SBA), silybin B (SBB), isosilybin A (ISBA), and isosilybin B (ISBB) [13,14].It exerts a diversity of biological and medical attributions including hepatoprotective, antioxidant, anti-cancer, antiviral and antibacterial properties.There are also reports on the protective role of SL against heavy metal toxicity [15][16][17][18].According to a recent report, silymarin could protect against Cd toxicity in human male spermatozoa [19].In another study investigating the deleterious effects of Cd on plant growth, silymarin has shown to improve photosynthesis e ciency and further increase the activity of various antioxidants [20].Silymarin and milk thistle have also shown to ameliorate toxic effects of Cd in male Japanese quail [21].
Despite promising results, poor absorbance, low aqueous solubility and permeability, and rapid metabolism of SL limits its therapeutic responses in clinic.An array of linking and encapsulation methods have been used to improve the physico-chemical properties of SL including complexation and nano-based delivery systems like micelles, liposomes, and phytosomes ets.Liposomes are spherical and colloidal carriers prepared from natural and synthetic phospholipids and cholesterol, which can improve the permeability of drugs through biological barriers [22].
Extensive data indicated that chronic exposure to inhaled Cd is associated with carcinogenesis, primarily in the lung as well as other smoking-related respiratory diseases, including pulmonary disease.Therefore, this study designed to investigate whether the nanoliposome form of silymarin (SL-L) could alleviate the toxicity of Cd on normal lung cells (MRC-5).Further, due to the higher sensitivity of lung cancer cells to Cd toxicity [4], different metabolic pathways and increased rate of toxic metabolites generation in cancer versus normal cells [23], we also compared SL-L protective effect against Cd toxicity in both MRC-5 and A 549 cancer cells.

Nanoliposome preparation and characterization
Liposomes were prepared using lipid lm hydration and extrusion method.Liposomal formulation were composed of phospholipids HSPC, cholesterol and DSPE-mPEG 2000 at (55:40:5) molar ratios.Dissolved lipids in chloroform were mixed in a round-bottom ask and the solvent was evaporated under vacuum in rotary evaporator (Heidolph, Germany) followed by lyophilization using lyophlizer (VD-800F, Taitech, Japan) for 2 h to remove traces of the remained solvent.The lipid lm was then hydrated with Histidine buffer (0.2 M, pH 7.0) and dispersed using a vortex at 50°C to form multi-lamellar vesicles (MLVs).The resulting MLVs were downsized by sonication (15 min) and extrusion through 200, 100 and 80 nm polycarbonate lters using a mini-extruder apparatus (Northern Lipids Inc., Canada).The liposomes were dialyzed against sucrose-phosphate (pH = 7) and were incubated with silymarin for 2 h at 60°C.The liposome preparation was dialyzed again in sucrose-phosphate (pH 7).The physical properties of liposomes; such as particle size and zeta potential, were determined by a particle size analyzer (Nano-ZS, Malvern Panalytical, Malvern, UK).In addition, the morphological features of liposomes were determined using transmission electron microscopy (TEM) (Zeiss, Jena, Germany).The entrapment e ciency of silymarin was calculated using Ultraviolet-visible (UV-Vis) spectrophotometry at 288 nm.Unencapsulated silymarin was removed by centrifugation.

Maintenance of cell lines
MRC-5 human broblast lung cells and A549 adenocarcinomic human alveolar basal epithelial cells were obtained from the Pasteur Institute of Iran (Tehran, Iran).Cells were cultured in DMEM HIGH GLUCOSE medium with 10% (v/v) fetal bovine serum, and 1% (v/v) penicillin and streptomycin.Cells were maintained at 37°C in a humidi ed atmosphere (90%) containing 5% CO2.

Cytotoxicity assay
First Cd cytotoxicity on MRC-5 (2.5, 5, 12.5, 25 and 50 µM) and A 549 (0.25, 0.5, 1, 2.5 and 5 µM) cells was determined using the methyl thiazol tetrazolium bromide (MTT) assay following 24 h incubation as described previously [24].Then, the toxicity of SL and SL-L (2.5, 5, 12.5, 25 and 50 µM) was determined on both cells using 24 h incubation time.To investigate the protective effect of SL and SL liposomes (SL-L) on Cd toxicity, MRC-5 and A 549 cell viability was evaluated using the same test.For this, MRC-5 cells were cultured in 96-well microtiter plates at 4× 10 3 cells/well.After 24 h incubation, cells were treated with various silymarin nanoliposomes concentrations of 2.5, 5 and 10 µM along with 25 µM Cd added in culture media.For A 549 cancer cells, 10 4 cells/well were seeded in 96-well microtiter plates.Following 24 h incubation time, cells were exposed to silymarin nanoliposomes concentrations of 2.5, 5 and 10 µ M with 0.25 µM Cd added in culture media.Both cells were also treated with free silymarin at 50 µM.Then, cells were incubated at 37°C for 24 h and the wells were washed with PBS and treated with MTT solution ( nal concentration 5 mg/mL) for 4 h in dark at 37°C.After incubation, the media was replaced with 200 µL dimethylsulfoxide (DMSO) to solve the purple formazan crystals.The absorbance of each palate was measured at 545 nm (630 nm as a reference) in an ELISA reader (Start Fax-2100, UK).All treatments were done in triplicate.The IC 50 values were determined using CalcuSyn version 2 software (BIOSOFT, UK).

Reactive oxygen species production assay
The generation of reactive oxygen species (ROS) was measured using cell-permeable uorescent probe CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′-dichlorodihydro uorescein diacetate acetyl ester).Upon intracellular hydrolysis by esterase, CM-H2DCFDA is transformed to a polar DCFH carboxylate anion, which is oxidized in the presence of intracellular ROS resulting in the generation of dichloro uorescein (DCF) with a strong uorometric emission.So, MRC-5 and A 549 cancer cells were seeded in 96-well plates at 4× 10 3 and 10 4 cells/well, respectively.After an overnight incubation at 37°C, cells were treated with SL and SL-L at 2.5, 5 and 10 µM along with 25 and 0.25 µM Cd added in culture media for MRC5 and A 549 cancer cells, respectively.Both cells were also treated with free silymarin at 50 µM.Following 48 h incubation, the cells were incubated with DCFH-DA (10 µM) for 30 min at 37°C.Fluorescence intensity was recorded at λem: 530 nm, λex: 485 micro plate uorimeter (Paradigm multi-mode plate reader, USA) [25].

Determination of apoptosis
Propidium iodide (PI) staining followed by ow cytometry was used to determine the anti-apoptotic effects of silymarin and silymarin liposome.Small fragments of DNA can be eluted following incubation in a hypotonic phosphate-citrate buffer.So, cells with less DNA contents will take up less DNA-binding color such as PI and will subsequently appear to the left of the G1 peak [26,27].So, MRC-5 and A 549 cells were seeded at 4× 10 4 and 10 5 cells/well in 12-well plates and incubated overnight at 37°C.Then cells were incubated for 24 hours with various concentrations of SL and SL-L at 2.5, 5 and 10 µM along with 25 and 0.25 µM Cd in culture media for MRC-5 and A 549 cells, respectively.
Both oating and adherent cells were collected and incubated over night at 4 • C in the dark with 200 µL of a hypotonic buffer (50 µg/ml PI in 0.1% sodium citrate and 0.1% Triton X-100) before ow cytometric analysis using a FACScan ow cytometer (Partec GmbH, Münster, Germany) [28].

Western blot
Western blotting was used to analyze the protein expression in MRC-5 and A 549 cells treated with SL-L as compared to the negative control.MRC-5 and A 549 cells (5×10 5 and 10 6 cells/ T75 ask, respectively) were treated with 10 and 2.5 µM silymarin liposome for 24 h, respectively.After washing twice with cold PBS, cells were resuspended in cold lysis buffer and incubated on ice for 30 min (Lysis buffer: 50 mM Tris-HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 10 mM NaF, 1 mM sodium orthovanadate (Na3VO4), 10 mM β glycerophosphate, 0.2% W/V sodium deoxycholate, 10 mM 2ME and 1% protease inhibitor cocktail and 1 mM phenylmethylsulfonyl uoride (PMSF).Then, the mixture was centrifuged at 4000 g for 10 min at 4 • C in a microcentrifuge.The supernatants were removed into chilled test tubes and the protein concentrations of each samples were determined using Bio-Rad Protein Assay kit.Each 20 µl of the total protein samples was loaded on 8% and 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (W/V) and then were transferred to PVDF membranes.Blocking of the membranes was done using 5% non-fat milk powder (skimmed milk) in Tris buffered saline tween 20 (TBST) on a rocker during 2 h at room temperature.The membranes were then incubated with primary antibodies including Caspase 3 (Cell Signaling, #9665, 1:1000), PARP (Cell Signaling, #9532, 1:1000) and Beta Actin (Cell Signaling, #3700, 1:1000) for 2 h at room temperature and were washed three times with TBST.Incubation continued with the appropriate anti-mouse IgG labeled with horseradish peroxidase (Cell Signaling, #7076, 1:3000) or anti-rabbit IgG labeled with horseradish peroxidase (Cell Signaling, #7074, 1:3000) for 1.5 h at room temperature.Caspase 3 protein band and PARP cleavage in both cells were detected by enhanced chemiluminescence using ECL western blotting detecting reagents.Images were quanti ed using Gel Doc UV Alliance, Alliance 4.7, UK [29].

Statistical analysis
Data were expressed as means ± SD of triplicate experiments and subjected to one-way ANOVA with subsequent followed by Tukey-Kramer.All analyses were performed using Graphpad Prism 8 software.Comparisons were made relative to untreated controls and signi cance of difference is indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.

Characterization of liposome
The liposome showed a good homogeneity, monomodal distribution con rmed by a low polydispersity index and the appropriate uniform size less than 150 nm (Fig. 1).The mean average diameter of nanoliposomes were 122.6 nm with zeta potential of -10.3.The negative zeta potential was due to the presence of mPEG 2000 -DSPE [30].The concentration of silymarin within liposomes was 1.2 mg/ml (1.2 mg of silymarin per ml of liposomal formulation).

The protective effect of silymarin liposomes on Cdinduced cytotoxicity
As mentioned earlier, we observed that silymarin liposome at concentrations above 25 µM signi cantly reduced MRC-5 and A 549 cell survival.Therefore, cells were incubated with different nontoxic concentrations of silymarin liposome (2.5, 5 and 10 µM), silymarin (50 µM) as well as Cd at either 25 or 0.25 µM in culture media for MRC-5 or A549 cells, respectively.NAC (6 µM) was also used as a strong antioxidant that interfere with the effect of Cd on the cells [32].After 24 h exposure, cytotoxicity was evaluated using MTT assay.All treatment groups could signi cantly improve MRC-5 cell survival compared to Cd (25 µM) (p < 0.001).As shown in Fig. 4A, a signi cantly increased MRC-5 cell survival was observed following SL-L treatment at 10 µM as compared to free silymarin (50 µM) (p < 0.05).Obviously, SL-L could enhance cell viability due to its antioxidant properties, which was further investigated in this study.SL-L e cacy was comparable to that of NAC, as a strong antioxidant (p > 0.05).Concerning A549 cells, treatment with SL-L at lower concentrations of 2.5 and 5 µM signi cantly increased cells viability compared to Cd (0.25 µM) (p < 0.001, p < 0.001, respectively)(Fig.4B).Further, SL-L at 2.5 µM was even more e cacious compared to silymarin (p < 0.05) and NAC (p < 0.01) in improving A 549 cell viability (Fig. 4B).It is implicated that phytochemicals pro-oxidant and anti-oxidant balance is concentration dependent.While SL-L at higher concentrations exhibit pro-oxidant and pro-apoptotic features in both cells, lower concentrations played a protective role on Cd induced toxicity.

The effect of silymarin nanoliposomes on ROS generation
To investigate the role of ROS in Cd toxicity, intracellular ROS levels were determined using DCF-DA uorescence staining assay in the cells.As shown in Fig. 5A, exposure of MRC-5 cells to free and liposomal silymarin or NAC signi cantly reduced ROS generation (p < 0.001).Silymarin liposome at 5 µM reduced ROS generation to a signi cant level compared to positive control (p < 0.001).Regarding A 549 cells, all treatment groups signi cantly reduced ROS levels compared to Cd treatment (p < 0.001) (Fig. 5B), however, silymarin liposome at 2.5 and 5 µM demonstrated a greater reduction in the ROS generation levels compared to NAC (p < 0.001).

Effects of silymarin and silymarin nanoliposomes on Cd-induced apoptosis
The proportion of apoptotic cells was determined following PI staining using ow cytometry as described previously [33].Results indicated the presence of a high proportion of the cell population in sub-G1 area of the peaks (a reliable biochemical markers of apoptosis) in Cd-exposed MRC-5 and A 549 cells when compared to untreated control cells (69.6% vs 5.17% and 67.5% vs 2.05%, respectively) (Fig. 6).However, the apoptosis rate of the cells treated with either free and liposomal silymarin or NAC showed a signi cant reduction.Following MRC-5 cells exposure to Cd at 25 µM, treatment with liposomal silymarin at either 2.5 or 5 µM signi cantly reduced the sub-G1 peaks compared to NAC (46.7% and 26.3% vs 63.8%).Similarly, treatment of A 549 cells with SL-L resulted in a reduced sub-G1 peak compared to NAC and free silymarin, following Cd exposure at 0.25 µM.While considerable reduction of the cell population in sub-G1 area was observed at 5 µM SL-L in MRC-5 cells, the A 549 cancer cells response was observed at lower concentration (2.5 µM liposomal silymarin) (26.3% for 5 µM in MRC-5 vs 27.7% for 2.5 µM in A 549) (Fig. 6).

Western blotting
To investigate the anti-apoptotic mechanism of silymarin nanoliposomes following Cd exposure in MRC-5 and A 549 cells, western blotting was used to measure the proteins levels involved in apoptosis including pro and cleaved caspase-3 as well as cleaved PARP.
As shown in Fig. 7A, with respect to A 549 cells, the cleaved caspase-3 protein showed a signi cant increase in Cd-exposed cells compared to control (p < 0.05), and NAC and silymarin nanoliposomes treatment signi cantly decreased cleaved caspase-3 protein expression (p < 0.001) compared to Cd.Further, silymarin nanoliposomes treatment was also more effective in decreasing cleaved caspase 3 compared to standard NAC treatment (p < 0.01) (Fig. 7A).In Cd-exposed MRC-5 cells, the levels of pro and cleaved caspase-3 showed a signi cant increase compared to control (p < 0.001), while SL-L at 10 µM signi cantly reduced pro and cleaved caspase-3 expression (p < 0.001) which was comparable to that of NAC effect (p > 0.05, respectively) (Fig. 7B).
In this experiment, western blot analysis was also done on PARP, a target of caspase and a marker of apoptotic cell death involved in DNA repair.Its cleavage onto fragments has been shown to be a hallmark of apoptosis [34].As shown in Fig. 8A, the levels of cleaved PARP signi cantly increased (p < 0.001) in Cd-exposed A 549 cells, and silymarin nanoliposomes at 2.5 µM could signi cantly decrease the PARP cleavage and inactivation (p < 0.01) which was also comparable to NAC results (p < 0.001).The same trend was observed regarding MRC-5 cells.PARP cleavage was signi cantly induced following Cd exposure in MRC-5 cells and silymarin nanoliposomes treatment at 10 µM signi cantly suppressed PARP cleavage (p < 0.001) which was similar to NAC effects (p < 0.001) (Fig. 8B).

Discussion
Heavy metals are known as serious toxicants and carcinogens, and environmental pollution from industrial and agricultural waste increase the risk of their exposure to human and animals [35,36].Cd toxicity has shown to be associated with apoptosis, oxidative stress, and DNA damage through various mechanism [37].In this study, we investigated the role of liposomal silymarin against Cd toxicity in MRC-5 human broblast lung cells as well as A 549 adenocarcinomic human alveolar basal epithelial cells.Our observation indicated that liposomal form of silymarin could signi cantly reduce Cd toxic effects on both cells and improve cell recovery following Cd exposure.
As previously reported, inhalation is the main route of Cd exposure and many lung diseases are attributed to the in ammation caused by Cd.The Cd retention in the lungs and other organs results in an impaired function of the host immunity and susceptibility to bacterial colonization and the subsequent chronic in ammation [38].Experimental investigation at cellular levels indicated that the up-regulation of several cytokines including IL-6 and MIP-2/CXCL2 in the human M1 broblasts and MIP-2, IL-1β and TNF-α in alveolar macrophages may have implications in the development of Cd-induced lung damage [39].Also, Cd at certain concentrations (20 to 60 µM) could increase the intracellular ROS levels in BEAS-2B human bronchial epithelial cells, resulting in the activation of apoptosis related pathways including JNK, ERK and p38 MAPK [40].
We compared the toxicity of Cd in both normal and cancer cells.Our results indicated that a signi cantly lower concentration of Cd could induce toxic effects in cancer cells compared to the normal cells.Further, we have shown that Cd exposure could signi cantly increase ROS generation and sub-G1 population in both cells.Results also indicated that Cd could increase the cleaved caspase 3 expression to a signi cant level in MRC-5 cells compared to A 549. Cd effect on ROS generation in C6 glioma cells has shown to be attributed to a Fenton-type reaction resulting in oxidative stress induction [41].Oh et all.have shown that ROS generation following Cd treatment could trigger apoptosis through caspase-dependent pathway including caspases 3, 8 and 9 in HepG2 cells [42].
Numerous studies indicated the protective roles of essential metals, vitamins, edible plants, phytochemicals, probiotics and other dietary supplements against Cd toxicity [43].Several reports also revealed direct competition of Zn with Cd for uptake through Zn transporters, calcium channels, and DMT1 (divalent metal transporter 1), however there are also other mechanisms including metallothionein induction and redox homeostasis [44].The role of oxidative stress following chronic Cd toxicity has long been demonstrated and it was shown that co-treatment with antioxidative agents could be promising [45].Chelating agents and their combination with antioxidants including ascorbic acid, alpha-tocopherol, and selenium have shown to protect against Cd toxicity in experimental animals [46][47][48][49].
In recent years, numerous studies have demonstrated the use of medicinal herbs as a potential treatment for detoxi cation of heavy metals due to the clearly fewer adverse reactions compared to chemical chelators [50].Silymarin (SL), a polyphenolic avonoid known for its promising pharmacological activities has received tremendous attention over the last decades.The antioxidant features of SL play a central role in its protective actions including direct scavenging of free radicals and chelating free elements, maintaining cellular redox balance, inhibiting ROS-producing enzymes, improving the integrity of mitochondria in stress conditions and decreasing in ammation responses [51].Various experimental models have also shown the antioxidant effect of free SL against drugs adverse reactions as well as toxicants including arsenic [52,53] and manganes [18,54,55].It's been shown that the protective effects attributed to silymarin might arise from its ability to trap free radicals and its chelating property including ferrous ions chelating activities [56].
The absence of ionizable groups and low aqueous solubility however, negatively affect silymarin bioavailability and its penetration through the biological barriers.These shortcomings could be modulated using nanotechnology-based drug delivery systems by improving SL solubility and penetration properties.In the past two decades, there have been exciting progress in the eld of nanomedicine, outlining nanoparticle capabilities for effective cellular interaction and subcellular targeting.Among various promising drug delivery systems, liposomes represent an advanced technology that paved the way to the clinic.The key feature of liposome is addressing two main issues in drug therapy including improving the stability and physicochemical properties of the entrapped agent as well as targeting the in ammatory tissues [57].
Our data indicated that higher concentrations of silymarin liposome (above 25 µM) exerted a considerable toxicity in both cells, while lower concentrations (10 and 2.5 µM for MRC-5 and A 549 cancer cells, respectively) exhibited a protective and anti-oxidant role in both cells.Indeed, SL-L at certain concentrations was effective in improving cell viability following Cd exposure in both A549 and MRC-5 cells, with comparable results to that of NAC as a well-known antioxidant [32].These results clearly clarify a concentration-dependent pro-oxidant and anti-oxidant feature attributed to the liposomal form of silymarin.Further, SL-L at lower concentrations was capable of improving the viability of A549 cell compared to MRC-5 cells.We also examined the protective effects of SL-L on the apoptosis induction and we have observed the same trend.Treatment with SL-L increased the sub-G1 population of both cells exposed to Cd, however, lower concentrations of SL-L was required to reduce the aforementioned cell population in A 549 cells (2.5 µM) compared to MRC-5 cells (5 µM).
The intrinsic apoptotic pathway of cell death is directed by caspases-9 and − 3 and is activated by different types of cell stress.There are reports indicating that caspase-3 inhibition could protect against the oxidative stress induced by Cd, thus suggesting caspase-3 activation as a major contributor to the generation of reactive oxygen species (ROS) [58].Our study demonstrated that SL-L treatment could reduce the cleaved caspase 3 protein expression in both cells.We have also shown that signi cantly lower concentrations of SL-L is required to exert the same effects in A 549 cancer cells compared to MRC-5 cells.There are however con icting results in the literature, for example, Shih et al. have failed to show the activation of pro-caspase-3 and cleavage of PARP following Cd exposure and concluded that Cd mechanism of toxicity is attributed to the mitochondria-mediated AIF translocation into the nucleus [59].The PARP protein is also an important downstream molecule in the apoptotic pathway and is thought to be a marker of caspase-3 protein activation of cells undergoing apoptosis [60].Our data demonstrated that treatment with SL-L could obviously decrease the PARP cleavage and the subsequent inactivation following Cd exposure in both cells.
The protective role of free SL against Cd toxicity was previously reported in several experimental animal models.For example, silymarin and milk thistle have shown to reduce the toxic effects of Cd in male Japanese quail [61].Additionally, SL reversed Cd toxicity by inhibiting lipid peroxidation and improving the total antioxidant power in adult mice [62].Herein, we report for the rst time that the liposomal form of silymarin could exert both pro-and anti-oxidant effects on the cells and that the concentration-dependent antioxidant properties could protect both normal and cancerous cells against Cd toxicity.However, obviously lower concentrations are effective in protecting A 549 cancer cells compared to MRC-5 normal cells.
Though there are no clear evidence on the concentration-dependent effects of polyphenolic compounds in normal versus cancer cells, it is likely that due to some unknown mechanisms the polyphenolic compounds exerts their antioxidants effects at lower concentrations in cancer cells.Another possibility might be the enhanced uptake by the actively proliferating cancer cells to meet their higher demands of nutrient requirement.Indeed, the nutrient uptake in normal cells is a well-organized process, which accelerates in abnormally high proliferative cancer cells [63].Previously, a study of pancreatic tumor xenograft model revealed a strikingly increased albumin uptake by the cancer cells to meet their high glutamine demand [64].There are also insights into the effect of polyphenols as a potent antioxidant, by scavenging ROS, chelating transition metals or upregulating antioxidant enzymes, or a prominent prooxidant through forming free radicals [65].For instance, though the anti-aging effects of resveratrol in age-related human diseases have been revealed, it's been shown that depends on the exposed concentrations and the type of the cells, resveratrol could exhibit pro-oxidant properties leading to oxidative DNA damage [66].Another example is evidence implicating the potential of green tea polyphenols in inducing ROS-mediated cancer cell death.Nevertheless, depending on the concentration of polyphenols and physiologic context of the interaction, it has been shown that polyphenolic compounds could scavenge ROS under conditions of high oxidative stress, thus preventing cell damage [48, 67-69].

Conclusion
There have been many reports of the positive roles of polyphenols in intoxications with Cd.We have shown that the activation of apoptotic pathway and the formation of reactive oxygen species (ROS) contributes to Cd toxicity in both MRC-5 and A 549. Also, we have shown that higher concentrations of silymarin liposome induce a considerable toxicity, while lower concentrations exhibited a protective and anti-oxidant role in both cells.Silymarin liposome at nontoxic concentrations inhibited Cd cytotoxicity, which might be explained by either the chelating properties of silymarin or preventing the reactive oxygen species generation and apoptosis inhibition.These results obviously indicate a concentration-dependent pro-oxidant and anti-oxidant feature attributed to the liposomal form of silymarin.Further, SL-L protective role on A549 cancer cells required lower concentrations compared to MRC-5 cells.This might be explained either by higher uptake of silymarin liposomes by the cancer cells or some unknown mechanism through which polyphenolic compounds exerts antioxidants effects at lower concentrations.In general, this report underlines the importance of liposomes delivery of low aqueous soluble polyphenolic compound to cross the biological membranes, as well as leveraging our understanding of the metal-induced dysregulation at cellular levels and the distinct physiopathological processes with cancer cells that might affect cellular internalization of nanomedicines.

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Figure 7 Levels
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