Mitigative DNA damage, whereas QRT-PCR was performed to evaluate

Mitigative Role of Garlic and Vitamin E against
Cytotoxic, Genotoxic, Apoptotic and Tumorigenic Effects of Lead Acetate and
Mercury Chloride on WI-38 Cells

Abstract

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Lead acetate
(Led) and mercury chloride (Mer) represent important ecological and public
health concerns due to their hazardous toxicities. Naturally found products
play a vital role as chemopreventive agent innovation. Objective: The current study aimed
to assess the modifying effect of garlic (Gar) and /or vitamin E (Vit E)
against the half-maximal inhibitory concentration (IC50) Led and / or Mer
induced cytotoxic, genotoxic, apoptotic and tumorigenic effects. Method:
Human lung
cells (WI -38) were pretreated with Gar and/or Vit E for 24 h, and then treated
with Led and/or Mer either alone or with their combination for 24 h. The MTT
assay was carried out to determine the
cytotoxicity of Led and Mer and the viability of Gar and Vit E. The alkaline
comet assay was used to assess DNA damage, whereas QRT-PCR was performed to
evaluate p53, pro-apoptotic (Bax) and anti-apoptotic (Bcl2) mRNA expression.

Results: Results of this study showed that IC50 of Led was (732.72µg/mL)
and for Mer was (885.83ug/mL), while cell viability effective dose for  Gar was (300 µg/mL) and for Vit  E was (26800 µg/mL).    Treating cells with the IC50 concentration
of  Led or Mer or their combination  using half IC50 of both of them induced
severe DNA damage. Bax expression was increased, while p53 and Bcl2 expressions
were decreased and an imbalance of Bax/Bcl2 ratio was occurred. Pretreatment of
cells with Gar and / or Vit E ameliorated the previous alternations.
Combination of Gar with Vit E exhibited the most protective effect. Conclusion: Led and Mer can induce
oxidative stress and change the expressions of apoptosis-related proteins in
WI-38 cells. Gar and Vit E may be promising protective candidate agent against
the toxic effect of heavy metals.

Keywords:
Garlic; Vitamin E; WI-38; Lead acetate; Mercury chloride; Genotoxicity;
Cytotoxicity.

1.     
Introduction

Heavy metals are relatively high density elements 1 that can induce
toxicity even at low doses 2. Recently, ecological and worldwide national
health care efforts are focused on the environmental contaminations caused by these
metals. Additionally, extensive use of
heavy metals in various
industries, as
agriculture and technology, has dramatically increased human exposure 3.

Exposure of
living organisms to lead and mercury dates back numerous
decades for several purposes 4.

Mercury is
found extensively in the biosphere and is disposed into the environment by
human activity for example; mining and through industrial waste. Mercury
chloride (Mer) is one of the essential constituents of the dental amalgam 5
and is a potent poison absorbed through the respiratory tract, digestive
system, and skin that causes many physiological and metabolic disorders in
humans and other animals 6. Previous studies have shown that Mer has
carcinogenic potential and can induce single-strand breaks, DNA damage, and a
dose-dependent increase in comet tail length even at low concentrations 6-8.

Humans are exposed to lead acetate (Led) through many sources;
air, water, food, and soil which are polluted via cigarette smoking, many
occupations, industry, lead mines, paints, and gasoline. Also, it is also used
in the manufacture of cosmetics. Absorption of Led through the gastrointestinal
tract is the main exposure pathway in children 9.  Led itself is genotoxic or improves the
effectiveness of other DNA-damaging agents 10. Led exposure, may stimulate
the formation of Reactive Oxygen Species (ROS) affecting free radical
scavenging enzymes and glutathione. Led toxicity originates from oxidative
stress; therefore, a therapeutic strategy to increase the anti-oxidant activity
of cells against Led poisoning is critical. Such a strategy aims to remove Led
from tissues (or to prevent its interactions with cellular macromolecules) and
supplements endogenous anti-oxidant molecules to provoke the cellular anti-oxidant
defenses. Chelating therapy is extensively used to treat Led toxicity with
agents like such as vitamins and thiol compounds that are well known to restore
various biochemical processes 11.

Chemopreventive mediators perform an essential
role in mitigating the hazardous effects of heavy metals. In this study, natural compounds from
different plants and vegetables were assessed for their ability to prevent and
treat various diseases.

Garlic oil (Allium sativum) (Gar) plays a vital role to protect
the body from many diseases; it enhances the immune system and acts as a
chemopreventive, anti-oxidant, and anti-microbial agent 12. Raji et al. 13
reported that the anti-oxidant property of Gar and its major organosulfur
constituents is contributed to its ability to scavenge H2O2
and to inhibit the chain of oxidation induced by a hydrophilic radical
initiation. Moreover, Asadaq  and Inamdar
14 showed that Gar regulates lipid levels in plasma that are deregulated as a
response to
Led and
Mer-intoxication.

Vitamin E (d-alpha Tocopheryl; Vit E) is well documented as the best
vital exogenous anti-oxidant that protects cells from various oxidative damages. The anti-oxidant
capability of Vit E may be correlated its efficient radical scavenging power 15, 16.

This work was planned to investigate the cytotoxic, genotoxic,
apoptotic, and tumorigenic effects of Led and/or Mer, and the possible
protective effect of Gar and/or Vit E through measuring mRNA expression of  p53, Bax and Bcl2 and DNA damage.

2.     
Materials and methods

2.1.
Cell lines

Human lung cells (WI-38) were obtained
from the Egyptian Company of Production of Vaccines, Sera and Drugs (Vacsera),
Cairo, Egypt.

2.2.Chemicals

 Lead 
acetate and Mercuric chloride 
were purchased from (Sigma- Aldrich Chemical Co., St. Louis, MO, USA);
dimethylsulfoxide (DMSO; Sigma, USA) ,Gar (Potent Garlic; Nutra Manufacturing,
USA) and Vit E (Natural E; Nutra Manufacturing, USA) .

2.3. Treatments

To determine the
half-maximal inhibitory concentration (IC50) of Led and Mer; the following
doses were diluted with DMSO; and tested: (2500, 1250, 625, 312.5, 156.25, and
87.12 µg/mL) and (2500, 1250, 625, 312.5, 156.25, 87.12, 39.06, and 19.53
µg/mL, respectively). In order to assess the maximum viability effect of Gar
and /or  Vit E; the tested doses were:
(300, 150, and 75 µg/mL) for Gar, (26800, 13400, and 6700 µg/mL) for Vit E, and
(300/26800, 150/13400, and 75/6700 µg/mL) for the combination of Gar/ and Vit
E.

To evaluate the
cytotoxic, genotoxic, and apoptotic effects and the migitative role of Gar and/or Vit
E, 13 treatment groups were assessed. In Group 1: WI-38 cells were incubated
without treatments as a control. Group 2: cells were treated with the IC50 of
Led (732.72 µg/mL). Group 3: cells were pretreated with the maximum viability
effective dose of Gar (300 µg/mL), then treated with the IC50 of Led. Group 4:
cells were pretreated with the maximum viability effective dose of Vit E (26800
µg/mL), then treated with the IC50 of Led. 
Group 5: cells were pretreated with Gar and Vit E  (300 and 26800 µg/mL; respectively), then
treated with the IC50 of Led; Group 6: cells were treated with Mer (885.83
µg/mL). Group 7: cells were pretreated with Gar (300 µg/mL), then treated with
the IC50 of Mer. Group 8: cells were pretreated with Vit E (26800 µg/mL), then
treated with the IC50 of Mer. Group 9: cells were pretreated with Gar and Vit
E  (300 and 26800 µg/mL; respectively),
then treated with the IC50 of Mer. Group 10: cells were treated with the
combination of half of IC50 of Led and Mer (366.36 and 442.915 µg/mL;
respectively). Group 11: cells were pretreated with Gar (300 µg/mL), then
treated with the combination of half of IC50 of Led and Mer. Group 12: cells
were pretreated with Vit E (26800 µg/mL), then treated with the combination of
half of IC50 of Led and Mer. Group 13: cells were pretreated with Gar and Vit
E  (300 and 26800 µg/mL; respectively),
then treated with the combination of half of IC50 of Led and Mer.

WI-38 cells were
pretreated with Gar and/or Vit E for 24 h, and then treated with Led and/or Mer
for 24 h for either Led or Mer alone or with their combination.  

2.4.Determination of cytotoxicity
of  Led and/ or Mer and the cell
viability effect of Gar and Vit E by MTT assay

Ninety-six well tissue culture plate were
inoculated with 1 x105 cells/mL (100 uL/well) and incubated at 37°C (24 h) to develop a complete
monolayer.  Growth medium was decanted
from 96-well micro titer plates after a confluent sheet of cells were formed, and
the cell monolayer was washed twice with wash media. Two-fold dilutions of
tested samples were made in RPMI medium with 2% serum (maintenance medium);  0.1 mL of each dilution was tested in
different wells leaving 3 wells as control, which received only maintenance
medium. Plates were incubated at 37°C and then examined. Cells were checked for any physical signs of
toxicity, including partial or complete loss of the monolayer, rounding,
shrinkage, or cell granulation, by using an inverted microscope. MTT solution
was prepared (5 mg/mL; in PBS) (BIO BASIC CANADA INC); 20 µL MTT solution was
added to each well and placed on a shaking table, 150 rpm (5 min), then was incubated
at (37?C, 5% CO2) for 1–5 h to allow the MTT to be
metabolized. The media was dumped off (and plates were dried on paper
towels to remove residue if necessary). Formazan (MTT metabolic product) was re-suspended
in 200 µL of DMSO and placed on a shaking table, 150 rpm (5 min), to thoroughly
mix the formazan into the solvent. Optical density was red (560 nm) and the
background was subtracted (620 nm). Optical density was considered directly
correlated with cell quantity 17.

2.5.         
 Determination of
apoptotic activity of Led and/or Mer using quantitative real-time polymerase
chain reaction (qRT-PCR) for analysis of pulmonary pro-apoptotic (Bax), Bcl2
gene family (Bcl2), and (p53) mRNA expression

2.5.1.     
Total RNA extraction

SV Total RNA
Isolation System (Promega, Madison, WI, USA) was used to extract total RNA from
cells pellets according to the instructions of the manufacturer. Concentrations
and purity of RNA were measured spectrophotometry.

2.5.2.     
Complementary DNA (cDNA)
synthesis

cDNA was
synthesized from 1 ?g of RNA using the SuperScript III First-Strand Synthesis
System as described in the manufacturer’s protocol (#K1621, Fermentas, Waltham,
MA, USA).

2.5.3.     
Real-time quantitative
PCR

Real-time PCR
amplification and analysis were done using an Applied Biosystems thermocycler
with software version 3.1 (StepOne™, USA). The reaction contained SYBR Green
Master Mix (Applied Biosystems). Gene-specific primer pairs ( Table 1)  were designed with Gene Runner Software
(Hasting Software, Inc., Hasting, NY, USA) from RNA sequences from the gene
bank. All primer sets had a calculated annealing temperature of 60°C.
Quantitative RT-PCR was performed in a 25-?L reaction volume consisting of 2X
SYBR Green PCR-Master Mix (Applied Biosystems), 900 nM of each primer, and 2 ?L
of cDNA. Amplification conditions were: 2 min at 50°C, 10 min at 95°C, 40
cycles of denaturation for 15 s, and annealing/extension at 60°C for 10 min.
Data from real-time assays were calculated using the v1·7 sequence detection
software from PE Biosystems (Foster City, CA, USA). Relative mRNA expression of
the studied genes was calculated using the comparative Ct method. All values
were normalized to ?-actin, which was used as the control housekeeping gene,
and reported as fold change over background levels detected in the diseased
groups 18.

2.6. Determination of genotoxicity
of Led and/or Mer by comet assay

Cellular
samples were transferred to 1 mL ice-cold PBS. The suspension was stirred for 5
min and filtered. Then 100 ?L of the suspension was mixed with 600 ?L of
low-melting agarose (0.8% in PBS); 100 ?L of the previous mixture was spread on
the pre-coated slides. Then coated slides were immersed in lysis-buffer (0.045
M Tris/borate/EDTA TBE, pH 8.4, containing 2.5% sodium dodecyl sulfate SDS)
for 15 min. Then slides were sited in an electrophoresis chamber with the same
TBE buffer, but without SDS. The electrophoresis conditions were adjusted to 2
V/cm for 2 min and 100 mA. Staining with ethidium bromide (EtBr; 20 ?g/mL) at
4°C was done. With the samples still humid, the DNA-fragment migration patterns
of 100 cells for each dose level were assessed with a fluorescence-microscope
(with excitation filter 420–490 nm). Comet tail lengths were measured from the
middle of the nucleus to the end of the tail with 40× magnification to count
and measure the comet. For visualization of DNA damage, we observed
EtBr-stained DNA using a 40× objective on a fluorescence-microscope.

Komet 5 image analysis
software developed by Kinetic Imaging, Ltd was used to quantify single cell gel
electrophoresis (SCGE) data (Liverpool, UK). A CCD camera was used to evaluate
the quantitative and qualitative extent of DNA-damage in the cells by measuring
the length of DNA migration and the percentage of migrated DNA. Tail moment was
calculated via the program 19.

2.7. Statistical analysis 

Data were expressed as
means ± SEM. The results were analyzed statistically by one-way
analysis of variance (ANOVA) using SPSS (Statistical Package for the Social
Sciences, version 16.0.1, Chicago, IL, USA) software. The levels of
significance were set at p? 0.05, p ? 0.01 and p? 0.001.

3.     
Results

3.1.
Morphological changes in WI-38 cells

Normal control cells were small and
spindle-like in shape, with clear and continuous edges. Different
concentrations of Led and/or Mer showed a reduction in the number of cells
per counted area. Many of the treated cells were enlarged and vacuolated, while
others became rounded. Mer induced granule formation in WI-38 cells. Groups 3:5,
7:9, and 11:13 there were no morphological changes.

3.2.
Cytotoxicity of Led or Mer on WI-38 cells

Different doses of either Led or Mer
significantly suppressed the proliferation of WI-38 cells in a dose-dependent
manner (p ? 0.001) compared with control cells (Fig. 1A, B).

3.3. Viability
effects of Gar and / or Vit E on WI-38 cells

There was a positive correlation between the dose of Gar and/or
Vit E and the viability of the cells. This indicates that Gar and/or Vit E
enhanced fibroblast proliferation (Fig. 1C, D, E).

3.4. Protective
power of Gar and / or Vit E against the cytotoxicity of Led or/and Mer (Fig. 2)

In groups 2 and 6, there
were significant growth inhibition of WI-38 cells (p ? 0.001) compared with
group 1. Treatment with the combination in group 10 significantly reduced the
number of cells compared with group 1 (p ? 0.01) (Fig. 2).

Groups 3:5, 7:9, and
11:13 showed significant enhancement of WI-38 proliferation (p ? 0.001) compared with
their relative non-pretreated groups (2, 6, and 10) (Fig. 2). Gar or Vit E
significantly diminished the toxic effect of the combination of Led and Mer in
groups 11 and 12 compared with groups 1, 2, and 6.

Gar significantly
increased the proliferation of the cells in group 11 compared with group 10 (p ? 0.05).

The most protective
effect on the cytotoxicity of Led and/or Mer was achieved by pretreatment of
WI-38 cells with the combination of both Gar and Vit E (groups 5, 9, and 13).

3.5.  Genotoxicity
of Led or/and Mer by Comet assay

Led and/or Mer treatments (groups 2 and 5)
led to a significant and dramatic increase in DNA damage (p ? 0.001), as
indicated by the length of the comet tail and the tail moment compared with
their corresponding values in group 1, as shown in Table 2 and Figure 3.

Gar and/or Vit E significantly reduced the
DNA-damage (p ? 0.001) in WI-38 cells. Protection with Gar only was more
effective than with Vit E only, while pretreatment with the combination of Gar
and Vit E was the most effective.

3.6. Analysis
of  p53, Bax and Bcl2 mRNA expression

Bax expression levels in
groups 2, 5, and 10 were significantly higher than those in group 1 (p < 0.001). Pretreatment of cells with Gar or Vit E either alone or in combination (groups 3:5, 7:9, and 11:13) produced a significant reduction of these levels compared with groups 1, 2, 5, and 10 (Fig. 4). The p53 and Bcl2 expression levels were significantly reduced in groups 2, 5, and 10 compared with those in group 1 (p < 0.001) (Fig. 4). The combination of Gar and Vit E (groups 5, 9, and 13) showed the most protective effect against Led and Mer-toxicity compared to groups 3, 4, 7, 8, 11, and 12 (Fig. 4). 4.      Discussion The present study addresses the induced cytotoxic, genotoxic, and apoptotic effects of Led and/or Mer, and the protective role of Gar and/or Vit E. Led and Mer-toxicity and their intoxication are well-documented and described pharmacological phenomena. It has been shown that Led and Mer induced oxidative stress, through which they caused cytotoxicity 20, 21. The results of this work revealed that cells treated with Led and/or Mer showed significant cytotoxicity. Vit E and/or Gar diminished the cytotoxicity of the studied heavy metals; this is consistent with the results of Adams and Salem and Salem 22, 23. It has also been shown that Gar interacts with cellular proteins and generates hydrogen sulfide, which directly scavenges ROS, and stimulates endogenous anti-oxidant defenses 24. Other investigators have shown that Vit E attenuates the release of ROS 25. Accordingly, Gar and Vit E undergo vital roles in the cellular protection against ROS. ROS can alter the signal transduction system to affect cell regulation and apoptosis 26. Apoptosis, a gene-regulated phenomenon, controls the rate of the cellular death and keeps a suitable quantity of cells in the body 27. It has been established that Led and Mer enhance the production of ROS and initiate apoptosis in different tissues and cells 28, 29, a vital mediator of their toxicity 28, 30-37. It has been indicated that both Led and Mer induced apoptosis in human cells 30, 38.39. In the current study, mRNA expression of Bax, Bcl2, and p53 were studied in cells treated with Led and/or Mer. Xu et al. 40 reported that these three proteins determine cell suicide. Bcl2 regulates the main apoptotic pathway (mitochondrial apoptotic pathway) 28, while Bax competes with Bcl2. In normal cells, Bax is found in the cytosol, but after the release of the signals of apoptosis, it changes its conformation and inserts into the outer membrane of the mitochondria 41. This enhances the opening of mitochondrial permeability transition-pores in the outer membrane, leading to cytochrome c release, caspase cascade activation, and finally DNA fragmentation causing cell death 42.  Furthermore, p53 controls Bax-mRNA expression, which plays a role in p53-mediated apoptosis. The Bax/Bcl2 ratio is essential for the determination whether cells will undergo apoptosis 43. Results of this study presented that Led and/or Mer significantly enhanced Bax-mRNA expression (p < 0.001). In contrast, Bcl2 and p53 mRNA expression were significantly reduced (p < 0.001), suggesting the involvement of apoptosis in this cytotoxicity. Moreover, Led and/or Mer increased the Bax/Bcl2 ratio, while pretreatment with the studied anti-oxidants decreased it, indicating that the apoptotic effect of Led and/or Mer can be prevented by the studied anti-oxidants.  DNA damage activates p53 which leads to apoptosis, thereby prevents the cell from accumulating functional damage to its DNA. Xu et al. 40 documented that p53 mRNA expression began to decrease when PC 12 cells (derived from a pheochromocytoma of the rat adrenal medulla) were treated with high doses of Led; this may explain the observation in this study revealed that Led and/or Mer decreased p53 mRNA expression. It may also be due to the severe cell damage caused by the selected doses. In the current study, the genotoxic effects of Led and/or Mer were studied using comet assay. DNA damage was induced by Led and/or Mer and the comet formation due to DNA fragmentation was observed. No DNA damage was observed in the untreated cells as the nucleus remained intact and no comet was formed. DNA damage was observed in the heavy metal-treated WI-38 cells as an increase in the length of the comet tail, and percentage of DNA in the tail. This suggests that both Led and Mer have genotoxic effects on WI-38 cells. Carey 44 indicated that abnormal DNA is a primary initiator of tumor growth and development. The equilibrium between lesion induction and repair determines DNA oxidation levels. Increased oxidative stress or decreased repair causes increased DNA lesions and mutation accumulation that initiates cancer. Accordingly, scavenging of radicals, including OH• and ROO•, is an effective approach in DNA damage prevention 45. Led may affect DNA directly via DNA structure destruction or indirectly by activation of caspases induced apoptosis 46. In vitro examination has shown that Led integrates with protein sulfhydryl groups and the DNA phosphate backbone 47. Led exposure may lead to DNA breakage associated with an alternate cellular-redox state and a significant downregulation of protein kinase C; this may explain how Led may promote tumor formation 48. The genotoxicity of Led may contribute to its capability to deter the rate of DNA synthesis and to affect DNA repairing processes 49, 50. Mer acts as a genotoxin through altering gene expression that affects cell survival and apoptosis 51. It is a reactive metal that has a high affinity for macromolecules and binds to DNA in vitro, affecting DNA structure 49, 52. De Flora et al. 53 suggested that Mer acts as a mitotic spindle inhibitor by attaching to SH-groups in eukaryotes. ROS produced via Mer enhances DNA damage, tumorigenicity, and carcinogenicity 54. Anti-oxidants may play a vital role in reducing the hazards of heavy metals 23, 55-56. Accordingly, the results of this work showed that pretreatment of WI-38 cells with Gar and/or Vit E protected the cells from the toxic and apoptotic effects of Led and/or Mer. Gar reveals anti-oxidant activity via scavenging ROS and activating cellular anti-oxidant enzymes 57. Ahn et al. 58 reported that Vit E has a chromanol ring with OH group that donate a hydrogen atom to decrease free radicals and a hydrophobic side chain allowing penetration into biological membranes. Here in, cells treated with Gar and Mer showed significant improvement of the studied parameters. These results correlate with the results of Massadeh et al., El-Shenawy and Hassan, and Kumar et al. 59-61 who reported that Gar administration may be an effective anti-oxidant treatment strategy for Led- and Mer-induced oxidative insult. Vit E protected both the liver and the kidney in vivo from Mer toxicity 62, 63. Moreover, Hamadouche et al. 64 demonstrated that Vit E-administration reduced Led genotoxicity and cytotoxicity in somatic and germ cells in rats, in accordance with the results of the current study. Conclusion This work may present possible pathways of Led and Mer toxicity and the promising protective effects of Gar and Vit E. First, both Led and Mer induced cell cytotoxicity via ROS production that induced DNA damage. Subsequently, they affected p53, Bax, and Bcl2 expression in response to DNA damage, leading to a disturbance in the Bax/Bcl2 ratio and finally apoptosis. Pretreatment of cells with Gar and/or Vit E mitigated the alteration of the studied parameters. This indicates that Gar and Vit E could be effective anti-oxidants against Led and/or Mer toxicity.