Mitigative Role of Garlic and Vitamin E against
Cytotoxic, Genotoxic, Apoptotic and Tumorigenic Effects of Lead Acetate and
Mercury Chloride on WI-38 Cells
(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:
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.
Garlic; Vitamin E; WI-38; Lead acetate; Mercury chloride; Genotoxicity;
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
agriculture and technology, has dramatically increased human exposure 3.
living organisms to lead and mercury dates back numerous
decades for several purposes 4.
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
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.
Materials and methods
Human lung cells (WI-38) were obtained
from the Egyptian Company of Production of Vaccines, Sera and Drugs (Vacsera),
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) .
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
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.
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
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.
Complementary DNA (cDNA)
synthesized from 1 ?g of RNA using the SuperScript III First-Strand Synthesis
System as described in the manufacturer’s protocol (#K1621, Fermentas, Waltham,
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
2.6. Determination of genotoxicity
of Led and/or Mer by comet assay
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.
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.
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).
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).
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.
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).
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.
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