Synthesis and biological evaluation of Piroxicam derivative as a lead chelator

Article abstract of DOI:10.1515/mgmc-2019-0008

Lead as a potent environmental and occupational pollutant, exerts its toxic effect mainly through oxidative stress induction. Currently, chelation therapy is the only medical management of metal intoxications in clinic, but its administration is associated with various side effects as well. In this study the protective effect of synthetized Piroxicam derivative was evaluated against lead toxicity in vitro. First the chelating activity of Piroxicam derivative was studied through Jobs method and ¹³C{¹H} NMR spectroscopy. Then the cytoprotective effect of Piroxicam derivative (10, 20, 50, 100 and 200 μg/mL) was evaluated and compared with that of EDTA (30 μg/mL) in the presence of lead nitrate (30 μg/mL). The EC50 value of Piroxicam derivative was calculated as well. Finally, the chelation efficacy and antioxidant effects of Piroxicam derivative in EC50 and 2EC50 values was assessed and compared with that of EDTA. Results showed that Piroxicam derivative chelates lead ion as much as EDTA. Moreover, Piroxicam derivative prevented lead-induced cells death more effectively than EDTA which is may due to its potent innate antioxidant activity. In conclusion, the synthetized Piroxicam derivative with possessing potent chelating activity as well as potent antioxidant activity, could be considered as potential drug target in management of toxic metals poisoning.

Full text of DOI:10.1515/mgmc-2019-0008

Main Group Met. Chem. 2019; 42: 73–80
Research Article
Open Access
Sayed Masoud Hosseini, Ali Imani, Milad Rahimzadegan, Saeid Mohammadi and
Alireza Golaghaei*
Synthesis and biological evaluation of Piroxicam
derivative as a lead chelator
Keywords: piroxicam derivative; chelator; toxic metal;
antioxidant
https://doi.org/10.1515/mgmc-2019-0008
Received June 18, 2018; accepted January 26, 2019.
Abstract: Lead as a potent environmental and occupati-
onal pollutant, exerts its toxic effect mainly through oxi-
dative stress induction. Currently, chelation therapy is the
only medical management of metal intoxications in clinic,
but its administration is associated with various side
effects as well. In this study the protective effect of syn-
thetized Piroxicam derivative was evaluated against lead
toxicity in vitro. First the chelating activity of Piroxicam
derivative was studied through Jobs method and 13C{1H}
NMR spectroscopy. Then the cytoprotective effect of
Piroxicam derivative (10, 20, 50, 100 and 200 μg/mL) was
evaluated and compared with that of EDTA (30 μg/mL) in
the presence of lead nitrate (30 μg/mL). The EC50 value of
Piroxicam derivative was calculated as well. Finally, the
chelation efficacy and antioxidant effects of Piroxicam
derivative in EC50 and 2EC50 values was assessed and
compared with that of EDTA. Results showed that Piroxi-
cam derivative chelates lead ion as much as EDTA. Moreo-
ver, Piroxicam derivative prevented lead-induced cells
death more effectively than EDTA which is may due to its
potent innate antioxidant activity. In conclusion, the syn-
thetized Piroxicam derivative with possessing potent che-
lating activity as well as potent antioxidant activity, could
be considered as potential drug target in management of
toxic metals poisoning.
1 Introduction
Lead (Pb) is a toxic heavy metal, which has many industrial
applications e.g. in batteries, cable sheaths, machinery
manufacturing, shipbuilding, light industry, ammunition
and other industries (Kennedy and Pennington, 2008).
At yearend 2017, total secondary refined lead production
was 1,010,000 t, moreover total secondary refined lead
production in December 2017 was 84,300 t, which was
3% more than that in December 2016 (Greenberg et al.,
2016). Thus widely lead distribution in the environment is
expected. Lead exposure could occur via inhaled air, dust,
food, or drinking water (White et al., 2007). Lead poisoning
usually occur chronically which can affect neurological,
hematological, cardiovascular, gastrointestinal, and renal
systems (Pfadenhauer et al., 2016). Oxidative stress is
common mechanism between toxic metals and almost all
of them caused oxidative damage, so that it is considered
as primary mechanism in pathogenesis of these agents.
There are two independent mechanisms by which lead
exerts toxic effects in living organism: (1) Direct generation
of reactive oxygen species (ROS) like as O2°, H2O2, and
hydroperoxides, (2) Depletion of antioxidants capacity in
cells. Delta-aminolevulinic acid dehydratase (ALAD) and
glutathione reductase (GR) are two enzymes inhibited
by lead. ALAD inhibition results in delta-ALAD elevation
which in turn enhances the formation of ROS. GR is an
enzyme responsible in reduction of oxidized glutathione
(GSSG) and formation of reduced glutathione (GSH). GSH
is a member of antioxidant defense system in cells. Thus
GR inhibition by lead results in GSH level reduction and
finally antioxidant defense system dysfunction (Ahamed
et al., 2005; Sugawara et al., 1991). These together cause an
imbalance between oxidative and antioxidative systems
of cells which results in over production of oxidative-free
radicals and associated reactive oxygen species. Alteration
of cellular proteins and lipids structure and function
* Corresponding author: Alireza Golaghaei, Department of
Pharmacology and Toxicology, School of Medicine, AJA University of
Medical Sciences, Tehran, Iran, e-mail: Golaghaei.md@gmail.com.
Sayed Masoud Hosseini, Department of Pharmacology and
Toxicology, School of Medicine, AJA University of Medical Sciences,
Tehran, Iran.
Ali Imani, Department of Medicinal Chemistry, School of Pharmacy,
Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Milad Rahimzadegan and Saeid Mohammadi, Department of
Pharmacology and Toxicology, School of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran.
Open Access. © 2019 Hosseini et al., published by De Gruyter.
Attribution alone 4.0 License.
This work is licensed under the Creative Commons
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S.M. Hosseini et al.: Piroxicam derivative as a lead chelator
is the main consequence of excessive ROS formation, and their capacity to change membrane fluidity (Lúcio
leading to cellular dysfunction in energy metabolism, cell et al., 2007). Thus, pharmacomodulation of anti-
signaling, cell cycle control, and overall biological activity inflammatory drugs is of interest in order to improve their
dysfunction which ends up in cellular death (Newsholme interactions with reactive oxygen species.
et al., 2016). Highly reactive species produced in oxidative
In this study the chelating activity of Pir derivative
stress can attack essential biomolecules like as lipids, (ethyl1,2-benzothiazoline-3-(2H)-one-2-acetate1,1-dioxide)
proteins, nucleic acids, and even cell structures, followed (Figure 1), was evaluated in vitro under lead exposure
by oxidative damages and many pathological processes taking EDTA as a positive control, since its chelating
(Van Antwerpen and Neve, 2004). Moreover, some activity has already been proven in previous studies.
other studies have shown that lead exposure result in Antioxidant activity of Pir derivative also was investigated
glutathione level decrease and malondialdehyde (MDA) through malondialdehyde (MDA) and the total glutathione
levels increase as a lipid peroxidation biomarker (Patrick, (GSH) content measurement. It is postulate that chelating
2006).
and antioxidative activity of Piroxicam derivative make it
The ideal medical management of metal intoxications a useful candidate in prevention and management of lead
is chelation therapy (Flora and Pachauri 2010), however or even any other toxic metal toxicity.
current chelating agents have various side effects which
limit their extensive clinical use (Wang et al., 2016). So
there is essential need to synthesis new chelator agent 2 Results
with high efficacy and low side effect.
Nonsteroidal anti-inflammatory drug (NSAIDs) are
2.1 Pir derivative is able to form metal
a drug class that reduce pain, decrease fever, prevent
complex with lead
blood clots and, in higher doses, decrease inflammation.
Piroxicam (Pir), as a potent NSAIDs, can forms stable
complex with metals like as Fe(II), Co(II), Ni(II), Cu(II)
and Zn(II) (Gehad and Nadia, 2004). Furthermore, anti-
inflammatory and antioxidant action of NSAIDs was
approved in previous studies (Petersen et al., 2008).
Reactive oxygen species production typically increased
during inflammatory process and it was shown that some
of anti-inflammatory drug, like as NSAIDs, have potential
to interact with reactive species and therefor prevent
oxidative damage. Previous studies showed that some
NSAIDs have potency to scavenge reactive oxygen species
and inhibit intracellular oxidase activity (Mouithys-
Mickalad et al., 2000). It was shown that NSAIDs,
especially oxicam compound, are reactive against ROS.
It was well documented that oxicam family of NSAIDs
have the scavenging capacity of hydroxyl radical (OH),
superoxide anion (O2−), hypochlorous acid (HOCl) and
hydrogen peroxide (H2O2). Structure/scavenging activity
relationships derived from H-NMR and C-NMR associated
with mass spectroscopy revealed that oxidation of the
C-3 carbon of the enolic functional group in oxicam
family is key mechanism in antioxidative activity of
these agents (Van Antwerpen et al., 2004). Although
studies demonstrate that oxicam family of NSAIDs exert
a significant scavengering activity and antioxidant effect,
they remain poorer scavengers as compared to thiol-
containing molecules (Van Antwerpen and Neve, 2004).
Moreover, studies revealed that antilipoperoxidation
The chelating activity of Pir derivative for lead was
evaluated using Job’s method based on the changes in the
UV-Vis absorption spectra due to the complex formation.
The result of complexation study indicates that Pir
derivative is able to form metal complex with lead and the
maximum ΔA (1.66) is occurred in 0.65 molar ratio (Figure
2). According to Job’s plot (ΔA versus molar ratio of ligand)
the stoichiometry of the complex is 2:1, in other word two
molecules of Pir derivative chelate one molecule of lead
ion. Furthermore, in the 13C{1H} NMR spectra of Pb-Pir
derivative we observed down field shifting in chemical
shifts of carbonyl functional groups compared to free
ligand (Figure 3).
activity of oxicams is due to their scavenging properties Figure 1:ꢀPir derivative structure.
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of Pir derivative in presence of lead. The black curve is the ΔA of Pir
derivative with the molar ratio of 0.65.
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Figure 4:ꢀCytoprotective effect of Pir derivative and EDTA against
lead nitrate-induced cytotoxicity in HepG2 cells. Cells were exposed
to growth medium alone (unexposed group) or growth medium
containing lead nitrate (30 μg/mL; negative control group) or growth
medium containing 30 μg/mL lead nitrate plus different non-toxic
concentrations of Pir derivative (10, 20, 50, 100 and 200 μg/mL) or
growth medium containing 30 μg/mL lead nitrate plus EDTA
(30 μg/mL; positive control group) for 48 h. ** p < 0.01 and
*** p < 0.001 compared with negative control group. # p < 0.05
compared with positive control group. Differences were compared by
one-way ANOVA. Data were expressed as mean SD of three identical
experiments made in three replicate.
200 μg/mL significantly increased, compared with
untreated group (negative control group) (p < 0.05,
p < 0.001 and p < 0.001 respectively). Pir derivative at
the dose of 100 μg/mL resulted in the highest protective
effect against lead nitrate cytotoxicity, compare with
lower doses. Furthermore, there was significant difference
between cytoprotective effect of Pir derivative (100 and
200 μg/mL) and EDTA (30 μg/mL) against lead nitrate
toxicity (p < 0.05). Cytoprotective effect of Pir derivative
at the dose of 100 and 200 μg/mL was comparable. Pir
derivative EC50 value calculated using prism software was
72 μg/mL (data not shown).
Figure 3:ꢀ13C{1H} NMR spectra of free Pir derivative (a) and Pb-Pir
derivative complex (b); down field shifting in chemical shift
of carbonyl groups (158.99 and 167.34 to 159.94 and 168.64
respectively). Chemical shifts of other carbon atoms within the
molecule remained unchanged.
2.2 Pir derivative increases cell viability in
the setting of lead exposure
Lead nitrate (30 μg/mL; 48 h incubation) caused
significant decrease in HepG2 cells viability by about
48%. The effects of different non-toxic concentrations 2.3 Pir derivative decreases lead uptake by
of the Pir derivative (10, 20, 50, 100 and 200 μg/mL) or
EDTA (30 μg/mL) on HepG2 cells viability in the setting
HepG2 cells
of lead nitrate exposure were shown in Figure 4. Our Co-treatment of lead nitrate (30 μg/mL) and EC50 or 2EC50
results show that higher doses of Pir derivative reduced value of Pir derivative resulted in lead uptake reduction
cytotoxicity of lead nitrate; the percentage of cell viability by HepG2 cells, compared with negative control group
in Pir derivative treated group at the dose of 50, 100 and (p < 0.05 and p < 0.001 respectively). Moreover, EDTA
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S.M. Hosseini et al.: Piroxicam derivative as a lead chelator
(30 μg/mL) and Pir derivative at the dose of 2EC50 had the of oxidative stress induced by lead, GSH content (the
highest and comparable efficacy in prevention of lead index of the intracellular non-enzymatic antioxidant
uptake by HepG2 cells; lead uptake reduced by about 60% defenses) was decreased and MDA level (biomarker for
in these group (Figure 5).
lipid peroxidation) was increased in exposed cells. These
consequences caused significant reduction of cell viability
in MTT test (Figure 4). These findings were in parallel with
previous reports suggesting that almost all toxic metal
caused oxidative damage (Mikirova et al., 2011; Patrick,
2006). Thus, compounds with both antioxidant and
chelating activity, could be effective candidate for toxic
metal poisoning.
Potential activity of oxicam compounds in biomedical
chemistry and their strong chelating activity for metal
ions make them to be of interest in recent investigations
(El-Gamel, 2009). UV-Vis and IR spectra data revealed that
Pir through amide–CO and pyridine-N, behaves as neutral
bidentate ligand coordinated to the metal ions (Gehad
and Nadia, 2004). Therefore, oxicam derivative through
bi-functional mechanism (chelating activity along with
antioxidant activity), could be a good candidate for drug
targeting in the setting of heavy metal toxicity, like as lead
toxicity.
2.4 Pir derivative reduces lipid peroxidation
and increases total glutathione content
Lead nitrate (30 μg/mL) caused significant increase in lipid
peroxidation and decrease in total glutathione content
compared to unexposed cells (cells exposed to growth
medium alone) (p < 0.001). EC50 value of Pir derivative
and 30 μg/mL EDTA decreased lipid peroxidation and
increased total glutathione content, compared with
untreated group (negative control group). The highest
reduction in lipid peroxidation and increase in total
glutathione content was observed in cells treated with
2EC50 value of Pir derivative (Figure 6).
3 Discussion
In this study Pir derivative (ethyl 1,2-benzothiazoline-
3-(2H)-one-2-acetate 1,1-dioxide) was synthetized and its
efficacy in lead chelation was assessed. Change in UV-Vis
and 13C{1H} NMR spectra of Pir derivative in the presence of
lead nitrate suggests its ability in complex formation with
lead. Moreover, the maximum ΔA of different molar ratio
of Pir derivative in presence of lead suggests the maximum
Pir derivative/lead complex formation which also provide
the stoichiometric ratio. In this experiment, the maximum
ΔA was observed in the molar ratio of 65:35 (65% Pir
derivative solution and 35% lead nitrate solution) (Figure
2) which suggests the requirement of two ligand molecules
(Pir derivative) for chelating of one lead ion. Another
strong evidence for chelating activity of Pir derivative
was provided by 13C{1H} NMR experiments. Down field
shifting observed in 13C{1H} NMR spectra is most likely due
to conjugation of carbonyl groups with positively charged
Pb2+. Afterward, the chelating activity of Pir derivative was
compared with that of EDTA through measurement of lead
uptake by HepG2 cells exposed to lead nitrate. The date
revealed that Pir derivative both in EC50 and in 2EC50 values
significantly reduced lead uptake percentage compared
with untreated cells. 2EC50 values of Pir derivative could
prevent lead uptake as much as EDTA (Figure 5). Although
lead chelation activity of Pir derivative and EDTA was
comparable, but surprisingly the percentage of viable
cells exposed to lead in Pir derivative treated group was
more than EDTA treated group (Figure 4). It assumed
that such potent cytoprotective effect of Pir derivative in
Cytotoxicity and oxidative damage induced by lead
exposure was observed here in HepG2 cells. As a result
1 5 0
1 0 0
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*
***
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5 0
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Figure 5:ꢀLead uptake percentage in Pir derivative and EDTA
treated group in ratio to negative control group. HepG2 cells were
exposed to growth medium containing lead nitrate (30 μg/mL;
negative control group) or growth medium containing 30 μg/mL lead
nitrate plus EC50 or 2EC50 values of Pir derivative or growth medium
containing 30 μg/mL lead nitrate plus EDTA (30 μg/mL; positive
control group) for 48 h. * p < 0.05 and *** p < 0.001 compared with
negative control group. Differences were compared by one-way
ANOVA. Data were expressed as mean SD of three identical
experiments made in three replicate. N.S: Not significant.
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1 0
8
lead induced oxidative damage, so that MDA levels were
decreased and GSH content was increased in treated
group compared with negative control group. Although
EDTA showed antioxidant effect, but it was considerably
less than that of 2EC50 value of Pir derivative. This could
also explain the difference between 2EC50 value treated
group and EDTA treated group in cell viability. In other
word, although Pir derivative and EDTA have comparable
chelating activity, but because of Pir derivative potent
innate antioxidant activity, the cell viability in Pir
derivative treated group was more compared with EDTA
treated group. The relatively weak antioxidant activity of
EDTA could be correlate to its lead chelating activity by
which inhibits lead induced oxidative stress.
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In conclusion, this study demonstrated that synthetized
Pir derivative could effectively chelate lead ion and its
chelating activity was comparable to EDTA. Moreover,
cytoprotective effect of Pir derivative against lead
exposure, was more than that of EDTA which probably is
due to its ROS scavenging properties and also its relatively
strong innate antioxidant activity. Since almost all toxic
metals share the same mechanism in oxidative stress
induction, synthetized Pir derivative may have protective
effect against any other toxic metal poisoning.
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Ethylchloroacetate,sodiumsaccharin,dimethylformamide,
Dimethylsulfoxide(DMSO)andEthylenediaminetetraacetic
acid (EDTA) were purchased from Merck (Germany)
Chemical Company. Lead nitrate (100% purity) was
obtained from Fisher Scientific in Fair Lawn, New Jersey.
Dulbeco’s Modified Eagle’s Medium (DMEM), fetal
bovine serum (FBS), penicillin and streptomycin, MTT
Figure 6:ꢀLipid peroxidation (A) and GSH (B) levels change in lead
nitrate exposed HepG2 cells after Pir derivative and EDTA treatment.
Cells were exposed to growth medium alone (unexposed group) or
growth medium containing 30 μg/mL lead nitrate (negative control
group) or growth medium containing 30 μg/mL lead nitrate plus
EC50 or 2EC50 values of Pir derivative or growth medium containing
30 μg/mL lead nitrate plus 30 μg/mL EDTA (positive control group) for
48 h. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with negative
control group. # p < 0.05 compared with positive control group.
Differences were compared by one-way ANOVA. Data were expressed
as mean SD of three identical experiments made in three replicate.
(3-(4, 5-dimethylthiazol-2-yl)-2,
5
diphenyltetrazolium
bromide), Bradford solution, thiobarbituric acid (TBA), and
5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) were purchased
from Sigma Chemical Company (St. Louis, MO, USA).
lead exposed cell is due to its potent innate antioxidant Synthesis of ethyl 1,2-benzothiazoline-
activity, compared with EDTA. For confirmation of this
assumption the antioxidant activity of Pir derivative and
3-(2H)-one-2-acetate 1,1-dioxide
EDTA was compared in cells exposed to lead (Figure 6). Synthesis of ethyl 1,2-benzothiazoline-3-(2H)-one-2-
According to data, both Pir derivative and EDTA reduced acetate 1,1-dioxide was done according to previous reports
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S.M. Hosseini et al.: Piroxicam derivative as a lead chelator
(D’Ascenzio et al., 2014; Jie et al., 2014). Ethyl chloroacetate solvent in our experiments. Chemical shifts for 13C{1H}
(11.9 g, 109.6 mmol) was added to a solution of sodium NMR spectra were referenced to the instrument’s locked
saccharin (14.5 g, 72 mmol) in dimethylformamide (25 mL). values for the solvent DMSO-d6 at 39.52 ppm.
The mixture was heated at 120°C for 3 h. The reaction
mixture was cooled to room temperature and then
poured into ice water (50 mL), resulting in an immediate
Cell culture
formation of white solid, which was filtered and washed
The human hepatocellular carcinoma (HepG2) cell line
with plenty of water. The solid was dried (70°C, overnight,
was purchased from National Center for Cell Sciences,
under vacuum) to produce (2) (17.6 g, 65.6 mmol, 85%) as
Pasteur Institute of Iran. In the laboratory, HepG2
white crystalline solid. m.p: 115-116°C; FT-IR (KBr) νmax
:
cells stored in liquid nitrogen. They were thawed by
immersion of their vials in a water bath at 30°C for
4 min. Afterward, the cells were transferred to a tissue
culture flask (75 cm2), diluted with DMEM containing
10% (v/v) fetal bovine serum (FBS) and 1% (v/v)
streptomycin and penicillin. Cells were maintained
in 5% CO2 incubator at 37°C. The culture medium was
renovated with fresh medium twice weekly. Cells were
detached by trypsinization when they reached 75-85%
confluency. Prior to the beginning of any biologic assay,
plates were changed to FBS-free medium. Because serum
might interfere with the results of assays. Furthermore,
it was shown that HepG2 cells have fairly good growth in
FBS-free DMEM (Alía et al., 2006).
1
2980, 1750, 1730, 1320, 1180 cm-1; H-NMR (400 MHz):
(CDCl3) δ: 8.38-7.90 (4H, m, aromatic), 4.60 (2H, s, CH2),
3.85 (2H, q, s, CH2), 1.12 (3H, t, CH3); LCMS (ESI): m/z: 270
[M+H], 292 [M+Na].
Complexation studies
The ability of Pir derivative to chelate the lead was
accomplished using the method of Job, in which the
complex formation caused the UV-Vis absorption maxima
intensity changes. In this method equimolar solutions of
metal ion and ligand are mixed and after recording the
UV spectra the difference in the maximum absorbance
(ΔA) versus the molar ratio (X) of the ligand is plotted;
in this way the stoichiometric coefficient of the complex Measurement of cell viability
can be achieved. In this study, 4×10-4 M solution of Pir
derivative in 1:5 DMSO/water and 4×10-4 M solution of For cell viability measurement, 200 µl growth medium
lead nitrate in water were prepared. The solutions of Pir containing 1 × 104 cells were seeded in each well of
derivative and lead nitrate were mixed in order to obtain 96-well plates, followed by an overnight incubation.
an increasing molar ration of Pir derivative (from 0.1 to After determination of non-toxic concentrations of Pir
0.95) and maintaining unchanged the total concentration derivative, cells were exposed to growth medium alone
of Pir derivative+lead nitrate (4×10-4 M). Each solution was (as unexposed group) or growth medium containing
placed in cuvette and the UV-Vis spectra were recorded lead nitrate (30 μg/mL (Yedjou and Tchounwou,
(Shimadzu uv-160A) in 223 nm (Pir derivative maximum 2007); as negative control group) or growth medium
absorbance) using an equimolar Pir derivative solution as containing 30 μg/mL lead nitrate plus different non-
blank. The stoichiometry of chelation can be calculated toxic concentrations of Pir derivative (10, 20, 50, 100 and
using following equation:
200 μg/mL; as treatment group) or growth medium
containing 30 μg/mL lead nitrate plus EDTA (100 µmol l-1 ≈
(1) 30 μg/mL (Fischer et al., 1998); as positive control group)
for 48 h (three replicates per experimental group). Cell
n = (1–X)/X
where “X” is the molar ratio that causes the maximum viability was determined using the MTT assay (Mosmann,
ΔA and “n” is the number of ligand molecules per cation 1983; Tchounwou et al., 2000). Briefly, supernatants were
present in the complex (Friggeri et al., 2015).
aspirated out and MTT solution was added to each well
13
We also performed C{1H} NMR studies of both free and incubated for 3 h at 37°C. Then, the supernatant was
Pir derivative and Pb-Pir derivative in accordance to replaced by isopropanol to dissolve the formazan crystals.
many similar works (Fang et al., 2014; Ye et al., 2002) on One hour after incubation with isopropanol, the formazan
synthesis of chelating compounds. For CNMR studies of dye was quantitated at 570 nm using an ELISA reader
Pb-Pir derivative we mixed lead nitrate and Pir derivative (Spectra Rainbow, Thermo, TECAN). The EC50 value of Pir
13
in 1:2 molar ratio. C{1H} NMR spectra were obtained on derivative was determined using GraphPad Prism v. 6.07
a BRUKER 400 MHz spectrometer. We used DMSO-d6 as software.
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S.M. Hosseini et al.: Piroxicam derivative as a lead chelator
ꢀ79
Lead uptake experiments
Pharmacology and Toxicology, School of Medicine, AJA
University of Medical Sciences, Tehran, Iran.
Cells were exposed to 30 μg/mL lead nitrate alone or
30 μg/mL lead nitrate plus EC50 or 2EC50 value of Pir
derivative or 30 μg/mL lead nitrate plus 30 μg/mL EDTA.
Following 48 h incubation at 37°C, the cells were washed
four times with PBS, detached by trypsinization and
aliquots counted. After sample preparation (Mikirova
et al., 2011; Tiffany-Castiglioni et al., 1996), lead contents
of cell were determined by atomic absorption spectrometry
(AAS; PERKIN-ELMER, 1100B). The lead content of the
samples was normalized with respect to cell numbers.
Conflict of interest: The authors declare no conflict of
interest.
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Lipid peroxidation assessment and total
glutathione content measurement
Cells were exposed to growth medium alone (unexposed
group) or growth medium containing 30 μg/mL lead
nitrate (negative control group) or growth medium
containing 30 μg/mL lead nitrate plus EC50 or 2EC50 values
of Pir derivative (treatment group) or growth medium
containing 30 μg/mL lead nitrate plus 30 μg/mL EDTA
(positive control group). Following 48 h incubation at
37°C, cell culture media were centrifuged at 1300 ×g
to separate any sedimentation. The cell pellets were
suspended in 400 µl chilled lysis buffer containing 250 mM
sucrose, 12 mM Tris-HCl, 0.1% Triton X-100, pH 7.4, 5 mM
PMSF and homogenized. The lysates were centrifuged at
12000 ×g for 10 min at 4°C and the cell extracts
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Statistics
Data were presented as mean standard deviation (SD).
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Differences were considered significant if P values were
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Acknowledgement: The study conducted under
supervision of Dr. Alireza golaghaei at Department of
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