chapter 3 in vitro radioprotective mechanism of...

Chapter 3 In vitro radioprotective mechanism of PMC-1

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Summary

The chapter deals with the in vitro radioprotective mechanism of protection of

quercetin-3-O-rutinoside (PMC-1), the major constituent isolated from PM. The

reaction of radiolytically generated hydroxyl radical with protein, lipid or DNA

molecules was prevented by PMC-1. Further, to understand the reactivity of these

radicals, pulse radiolysis studies were performed. The determined rate constants for

the reactions with different free radicals [hydroxyl (•OH) and trichloromethyl peroxyl

(CCl3O2•)] indicated antioxidant activity of PMC-1. The apoptotic effect of PMC-1

was further established by monitoring levels of phospho-p53, p53, Bad, cleaved

caspase-3 and cleaved PARP in irradiated V79 cells. PMC-1 pretreatment

significantly (p<0.05) decreased the radiation-induced increase in of phospho-p53,

p53, Bad, cleaved caspase-3 and cleaved PARP [Poly (ADP-ribose) polymerase] in

V79 cells. PMC-1, by itself did not produce any change in apoptotic markers compare

to normal control.

3.1. Introduction

The role of free radicals in disease manifestation has drawn considerable attention

over the past two decade. Free radicals can be defined as molecules (or molecular

fragments) with an unpaired electron in the outer orbital [1]. Due to the unpaired

electron, free radicals have high chemical reactivity and short half-life [2]. They can

react with macromolecules such as proteins, DNA, and lipids to produce various

damaging effects depending upon their reactivity [3].

To understand the reactivity of radicals which occur on timescales faster than

approximately 10-4-10-3 s, pulse radiolysis technique were used. Pulse radiolysis is a

method for studying events occurring between 10-11 and 10-2 s after energy absorption

[4]. This provides the extent and timescale of radiation damage in dilute aqueous

solutions and a simplistic use of experimental conditions to isolate a single reactive

species [5].


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As described in chapter 2, we isolated and characterized PMC-1, as the most potent

compound based on its antioxidant potential, protection against radiation-induced

ROS and DNA damage in V79 cells. Based on the results obtained from chapter 2, we

focused on evaluating the in vitro radioprotective mechanism of PMC-1.

3.2. Materials and Methods

3.2.1. Chemicals

2-propanol, Bovine serum albumin (BSA), carbon tetrachloride (CCl4),

dinitrophenylhyodrazine (DNPH) and guanidine hydrochloride were purchased from

Sigma Chemicals (St.Louis, MO, USA). BCA protein kit was purchased from Thermo

Fisher Scientific Inc., (MA, USA). Thiobarbituric acid (TBA) and trichloroacetic acid

(TCA) were purchased from HiMedia, Mumbai. Butylated hydroxytoluene (BHT)

was purchased from Merck, Mumbai. Plasmid DNA (pBR322) and loading buffer

were obtained from Bangalore Genei, India. All the other chemicals and solvents used

were of analytical grade. The reagent solutions were prepared in nanopure water from

a Millipore Milli-Q system just before the use.

3.2.2. Protein carbonylation assay

One milligram of protein was dissolved in 1 ml of 10mM phosphate buffer (pH 7.4).

Different concentrations of PMC-1 (0-100µM) were added to the protein solution.

Control sample was prepared by substituting test compound with buffer. Samples

were exposed to -radiation at an absorbed dose of 50 Gy (dose rate 40 Gy/min).

Following irradiation, the protein was precipitated with ice chilled 40% TCA. The

pellet was suspended in DNPH and incubated at room temperature for 4 h. Proteins

were reprecipitated with 10% TCA. Excess DNPH was removed by washing with


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ethyl acetate in ethanol (1:1) till the pellet decolorized. The protein pellet was then

dissolved in 6N guanidine hydrochloride and the absorbance was measured at 370

nm. The results are expressed in terms of percentage inhibition in radiation-induced

protein carbonylation [6].

3.3.3. Lipid peroxidation assay

Lipid peroxidation (LPO) studies were carried out in rat brain homogenate in

phosphate buffer (pH 7.4). Samples were exposed to -radiation at an absorbed dose

of 200 Gy (dose rate 40 Gy/min) both in the absence and presence of different

concentrations of PMC-1 (0- 100µM). The extent of LPO was estimated in terms of

thiobarbituric acid reactive substances (TBARS) using TBA reagent (15% w/v TCA,

0.375% w/v TBA, 0.25 N hydrochloric acid, 0.05% w/v BHT). The absorbance was

measured at 532 nm. The results are expressed in terms of percentage inhibition in

radiation-induced LPO [6].

3.2.4. pBR322 DNA damage assay

Agarose gel electrophoresis was employed to assess the radiation-induced damage to

pBR322 DNA. Agarose gel (1%) was prepared in 130 mM tris-borate/2.5 mM EDTA

(TBE) buffer, ethidium bromide was added to the gel (for visualization of the DNA

bands in a UV transilluminator). About 400 ng of pBR322 DNA was suspended in 20

l of 10 mM sodium phosphate buffer (pH 7.4) and exposed to an absorbed dose of

50 Gy (dose rate 40 Gy/min) both in the absence and presence of different

concentrations of PMC-1 (0-50 µM). Control and test samples were mixed with

loading dye (0.25% bromophenol blue and 30% glycerol) and loaded into the wells.

The gel was submerged in an electrophoresis tank filled with tris borate EDTA (TBE)


105

buffer. Electrophoresis was carried out at 60 V for one and half hours to separate the

open circular (OC) and the super coiled (SC) form of DNA. The movement of the

DNA bands was visualized on a UV transilluminator. The band intensity was

quantified by uvitec software (Gel documentation system) [6, 7].

3.2.5. Pulse radiolysis studies

3.2.5.1. Reaction of hydroxyl ( OH) and trichloromethyl peroxyl (CCl3O2 )

radical

Pulse radiolysis experiments were carried out with high-energy electron pulses (7

MeV, 500 ns) obtained from linear electron accelerator (LINAC) assembly at BARC,

Mumbai. [8]. The transients were detected by absorption spectrometry. The absorbed

dose was measured by using aerated thiocyanate dosimeter by monitoring the

(SCN)2 species at 475 nm with G value of 2.59 x 10-4 m2J-1 [9]. Here, G denotes the

radiation chemical yield in mol/J and , the molar absorption coefficient in m2/mol.

typical dose/pulse used for these studies varied from 8 Gy to 100 Gy. Radiolysis of

water produces e-aq, H and OH radicals in addition to the molecular products H2,

H2O2, and H3O+ [10, 11].

(1)

The reaction with OH radicals was carried out in the presence and absence of PMC-1

(30µM) in N2O-saturated solutions, where eaq- is quantitatively converted to OH

radicals

(2)

CCl3O2 radicals were generated by irradiating the aerated aqueous solution in the

presence and absence of PMC-1 (130µM) containing 48% 2-propanol, 4% of CCl4

2 aq 2N O e OH OH N

H2O H, OH, e-

aq, H2, H2O2, H3O+


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and 48% 20 mM phosphate buffer (pH = 8.5) [12, 13]. The formation of CCl3O2

radical was as follows.

(3)

(4)

Based on the above studies and results obtained from chapter 2, PMC-1 was further

taken up to understand the mechanism of action.

3.2.6. Mechanism of protection

3.2.6.1. Apoptosis assay in V79 cells by kit method

Cell culture conditions and Irradiation protocol are described in chapter 2; section

2.2.8.1 and 2.2.8.2.

3.2.6.1.1. Experimental design

Group 1 – Normal control

Group 2 – Drug control (PMC-1 alone)

Group 3 – Radiation control (10 Gy)

Group 4 – PMC-1 + Radiation: Cells treated with PMC-1 (50µM) for 30 min

prior to -radiation (10 Gy).

(CH3)2CHOH + H

/

OH (CH3)2C

OH + H2/H2O

(CH3)2C

OH + CCl4 CCl3 + (CH3)2CO + H+ + Cl

–

CCl4 + e–

aq CCl3 + Cl– (5)

CCl3

+ O2 CCl3O2 (6)


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3.2.6.1.2. Preparation of cell lysate

1. V79 cells were incubated with PMC-1 (50 M) for 30 min and then exposed

to -radiation (10 Gy).

2. After irradiation, cells were harvested under nondenaturing conditions,

medium was removed and cells were rinsed once with ice-cold phosphate

buffer saline (PBS).

3. PBS was removed and 0.5 ml ice-cold 1X cell lysis buffer plus 1mM

phenylmethylsulfonyl fluoride (PMSF) was added to each plate and incubated

on ice for 5 minutes.

4. Cells were scraped off the plate and transferred to appropriate tubes.

5. Lysates were sonicated on ice, microcentrifuged for 10 minutes at 4°C and

transferred the supernatant to a new tube.

6. The supernatant is the cell lysate. Single-use aliquots were stored at –80°C.

3.2.6.1.3. Total protein content

Total protein in cell lysate was estimated by the BCA Protein Assay Kit (Thermo

Fisher Scientific Inc., MA, USA). The protocol was followed by the standard

procedure supplied by the manufacturer of the kit as described in chapter 2.2.8.8.4.

3.2.6.1.4. Test Procedure

The method employed follows PathScan® Apoptosis Multi-Target Sandwich ELISA

Kit #7105 (Cell Signaling Technology Inc., Danvers, MA, USA). The protocol was

followed by the standard procedure supplied by the manufacturer of the kit as shown

below.


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1. 100 l of sample diluent (0.1% Tween 20 in 20 X PBS) is mixed with 100 l

of cell lysate in a microcentrifuge tube and then vortexed for a few seconds.

2. 100 l of each diluted cell lysate was added to the appropriate well, sealed

with tape and pressed firmly onto top of microwells. The plates were

incubated overnight at 4°C, which gives the best detection of target protein.

3. Gently removed the tape and washed the wells following the steps below.

a. Discarded plate contents into a receptacle.

b. Washed 4 times with 1X wash buffer, 200 l each time for each well.

c. After each wash, plates were tapped on fresh tissue papers to remove

the residual solution in each well, without allowing the wells to

completely dry.

d. Cleaned the underside of all wells with a lint-free tissue.

4. 100 l of detection antibody (green-colored bottle matching with the

corresponding marker) was added to each well, sealed with tape and incubated

the plate for 1 hour at 37°C.

5. Repeated wash procedure as in Step 3.

6. 100 l of horseradish peroxidase (HRP)-linked secondary antibody (red-

colored solution) was added to each well, sealed with tape and incubated the

plate for 30 minutes at 37°C.

7. Repeated wash procedure as in Step 3.

8. 100 l of 3,3 ,5,5 -tetramethylbenzidine (TMB) substrate was added to each

well, sealed with tape and incubated the plate for 30 minutes at 25°C.

9. 100 l of STOP solution was added to each well and shook gently for a few

seconds.


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10. Absorbance of each well was read at 450 nm using an ELISA microplate

reader and following markers were assessed; phospho-p53, p53, Bad,

phospho-Bad, cleaved caspase-3 and cleaved PARP.

3.2.7. Statistical analysis

Statistical significance between the groups was determined by one-way analysis of

variance (ANOVA) followed by post hoc Tukey’s test using GraphPad Prism version

5.02. p < 0.05 was considered to be significant. All values were expressed as mean ±

standard error of mean (SEM).

3.3. Results

3.3.1. Inhibition of radiation-induced protein carbonylation and lipid

peroxidation by PMC-1

Fig. 3.1 (A & B) represents percentage inhibition of protein carbonylation and lipid

peroxidation with increasing concentration of PMC-1. IC50 values for protein

carbonylation and lipid peroxidation were found to be 51.1 ± 1.9µM and 42.5 ± 1.2

µM respectively.


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Fig. 3.1. % inhibition of protein carbonylation (A) and LPO (B) with varying

concentrations of PMC-1. All values are expressed as mean ± SEM and experiments

were carried out in triplicate.

3.3.2. Inhibition of radiation-induced pBR322 DNA damage by PMC-1

Fig. 3.2 shows agarose gel electrophoresis pattern. Lanes 1-6 show the pattern of

separation for the irradiated plasmid DNA in the presence/absence of varying

concentrations of PMC-1 (0-50 µM). On exposure to -radiation, the SC form of

DNA was converted to the OC form. In presence of PMC-1, the damage to the SC

form was reduced. The intensities of OC and SC form of plasmid DNA were

measured and the ratio of OC/SC was plotted as a function of the concentrations of

PMC-1 (Fig. 3.3). IC50 values were estimated to be 25.8 ± 2.5 µM.

0 25 50 75 1000

20

40

60

80

IC50 = 51.1 ± 1.9 µM

PMC-1A

Conc (µM)

% in

hibi

tion

of p

rote

inca

rbon

ylat

ion

0 25 50 75 1000

20

40

60

80

IC50 = 42.5 ± 1.2 µM

PMC-1

Conc (µM)

% in

hibi

tion

of L

PO

B


111

Fig 3.2. Agarose gel electrophoretic pattern, depicting super coiled and nicked open

circular forms of plasmid DNA protection, by varying concentrations of PMC-1

against 50 Gy -radiation induced strand breaks. Electrophoresis was carried out at 60

V for 90 min to separate the open circular (OC) and the super coiled (SC) form of

DNA. The arrow indicates the direction of electrophoretic mobility.

Lane 1: pBR322 control

Lane 2: pBR322 treated with PMC-1 alone

Lane 3: pBR322 exposed to -radiation of 50 Gy

Lanes 4-6 : pBR322 exposed to -radiation dose of 50 Gy in presence of 10, 25 and

50 M of PMC-1 respectively

0 10 20 30 40 500

20

40

60

80

IC50 = 25.8 ± 2.5 µM

PMC-1

Conc (µM)

Rat

io o

f OC

/ SC

pB

R32

2

Fig. 3.3. Line graph showing ratio of OC/SC form as a function of the concentrations

of PMC-1.


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3.3.3. Pulse radiolysis

3.3.3.1. Reaction of hydroxyl radical ( OH) with PMC-1

Pulse radiolysis experiments revealed that the reaction of OH radical with PMC-1

(30µM) produced a transient which has absorption maxima at 470 nm, and a

bleaching signal at 390 nm, due to the parent absorption. The transient spectra for the

reaction of OH radical with PMC-1 are shown in Fig. 3.4.

Figure 3.4. Transient spectrum obtained during reactions of OH radical with

PMC-1.

The reaction of OH radical with aromatic substrate proceeds through different ways,

such as addition to ring, one-electron oxidation and hydrogen atom abstraction. To

understand the mode of reaction for OH radical, the transient spectra with specific

one electron oxidant, azide radical (N3 ) was carried out.

The transient spectra for the reaction of N3 radical with PMC-1 are shown in Fig. 3.5

which also has absorption band at 470 nm and bleaching at 390 nm. By comparing


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both the spectra obtained from OH radical and N3 it is evident that the species

absorbing at 470 nm was due to one electron oxidized product of PMC-1 that is the

corresponding phenoxyl radical derived from phenolic OH groups present in the

molecules.

The phenoxyl radicals thus produced are easily stabilized throughout the flavan

nucleus. The bimolecular rate constant for the reaction of N3 radical with PMC-1 was

determined from the buildup kinetics of the transient at 470 nm in presence of

different concentration of PMC-1. The bimolecular rate constant (k) was determined

from the slope of linear regression plot of solute concentration against kobs using

equation 1, as shown in Fig. 3.5.

[ ]obsk k solute (1)

The bimolecular rate constant of PMC-1 for the reaction with N3 radical was

estimated as 4.15 x 109 M-1s-1.

300 350 400 450 500 550 600

-0.04

-0.03

-0.02

-0.01

0.00

0.01

0.02

0.03

0.04

25 50 75 100 1250.0

2.0

4.0

6.0

k obs/1

05 , s-1

Abs

orba

nce

Wavelength, nm

4 s 8 s 20 s 40 s

[PMC-1], M

Figure 3.5. Transient spectrum obtained during reaction of N3 radical with PMC-1.

Inset shows the slope of linear regression plot of PMC-1 concentration against kobs.


114

3.3.3.2. Reaction of trichloro methyl peroxyl radical (CCl3O2 ) with PMC-1

Peroxyl radicals are one of the common free radicals produced during oxidative

stress. Halogenated peroxyl radicals are model peroxyl radicals, commonly used to

study the reaction with antioxidants or free radical scavengers. Fig. 3.6 shows the

transient absorption spectra of PMC-1 after reaction of CCl3O2 radical at pH 7.

The transient formed by CCl3O2 radical reaction with PMC-1 has weak broad

absorption band from 470 nm along with a bleaching signal at 380 nm. The reaction

of CCl3O2 radical with PMC-1 was relatively slower than that of OH radical because

CCl3O2 radical is less powerful oxidant than OH radical. As discussed earlier for

OH and N3 radicals, the band at 470 nm in Fig. 3.6 is assigned for oxidation of

PMC-1 by CCl3O2 radical.

The bimolecular rate constant of PMC-1 for the reaction with CCl3O2 radical was

estimated as 3.47 x 107 M-1s-1 using equation 1.

350 400 450 500 550 600

-0.015

-0.010

-0.005

0.000

0.005

0.010

60 90 120 1506.0

7.0

8.0

9.0

k obs/1

03 , s-1

Abs

orba

nce

Wavelength, nm

50 s 100 s 250 s 430 s

[PMC-1], M

Fig. 3.6. Transient spectrum obtained during reactions of CCl3O2 radical with PMC-

1. Inset shows the slope of linear regression plot of PMC-1 concentration against kobs.


115

3.3.4. Mechanism of protection

3.3.4.1. Evaluation of apoptotic markers in irradiated V79 cells

Fig. 3.7 shows -radiation-induced changes in apoptotic markers in V79 cells. PMC-1

pretreatment significantly (p<0.05) decreased the radiation-induced increase in

phospho-p53 (2.2 fold), p53 (2.0 fold), Bad (1.6 fold), cleaved caspase-3 (1.4 fold)

and cleaved PARP (1.6 fold) in V79 cells, compared to radiation control.

However, there was no significant change observed in radiation-induced phospho-Bad

level in V79 cells. PMC-1, itself did not produce any change in apoptotic markers

compare to normal control.


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0

20

40

60

80

Normal controlPMC-1 controlRadiation controlAPMC-1 + radiation

*

#

p53

activ

ity p

erg

of p

rote

in

0

20

40

60

80

Normal controlPMC-1 controlRadiation controlBPMC-1 + radiation

*

#

Phos

pho-

p53

act

ivity

per

g of

pro

tein

0

20

40

60

80

Normal controlPMC-1 controlRadiation controlCPMC-1 + radiation

*

#

BA

D a

ctiv

ity p

erg

of p

rote

in

0

20

40

60

Normal controlPMC-1 controlRadiation controlDPMC-1 + radiation

Pho

spho

-BA

D a

ctiv

ity p

erg

of p

rote

in

0

20

40

60

80Normal controlPMC-1 controlRadiation control

E

PMC-1 + radiation

*

#

Cle

aved

cas

pase

-3 a

ctiv

ity p

erg

of p

rote

in

0

20

40

60

80

Normal controlPMC-1 controlRadiation control

F

PMC-1 + radiation

*

#

Cle

aved

PA

RP

activ

ity p

erg

of p

rote

in

Fig. 3.7. Effect of PMC-1 on apoptotic markers in irradiated (10 Gy) V79 cells. (A)

p53, (B), phospho-p53, (C) Bad, (D) phospho-BAD, (E) cleaved caspase-3 and (F)

cleaved PARP. Results are presented as mean ± SEM (n=3). *p < 0.05 as compared to

normal control, #p < 0.05 as compared to radiation control.


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Discussion

Free radicals, generated by radiation, attack bio-molecules such as the fatty acid

component of membrane lipids and DNA, leading to lipid peroxidation, stand breaks

and ultimately cell death [14]. The reaction of radiolytically generated hydroxyl

radical with protein, lipid or DNA molecules was prevented by PMC-1. On exposure

to -radiation, the SC form of DNA was converted to the OC form. In presence of

PMC-1, the damage to the SC form was reduced. This indicates that radiation-induced

DNA damage which is primarily mediated by hydroxyl radicals is attenuated

effectively by PMC-1. Further, pulse radiolysis studies of PMC-1 were performed to

determine rate constants towards reactions with different free radicals, indicating

radical scavenging property of PMC-1. The ability of PMC-1 to inactivate these

radicals may be attributed to the presence of phenolic OH group. The above activities

may be probably due to 3’,4’-catechol ortho-dihydroxy moiety present in the B-ring

of PMC-1, which has a strong affinity for phospholipid membranes as well as due to

the presence of multiple hydroxyl groups that make these molecules excellent free

radical scavengers [15, 16]. Additionally, oxidation of B ring present in the catechol

structure of PMC-1 results the formation of an ortho-semiquinone radical, which has

been reported to be involved in the anti-lipid peroxidation activity [17].Therefore, the

strong antioxidant potential probably depends on the structure of PMC-1, especially

the number and arrangement of hydroxyl groups and the extended conjugation in the

molecular structure.

Another important aspect of radioprotective potential of PMC-1 at cellular level is its

ability to modulate the cytoprotective and cytotoxic pathways. V79 cells have been

widely used in the studies of DNA damage and DNA repair [18]. Radiation treated

V79 cells showed increased levels of phospho-p53, p53, BAD, cleaved caspase-3 and


118

cleaved PARP, indicating apoptosis. Apoptosis occurs either through the intrinsic

mitochondrial or extrinsic death receptor pathway [19, 20]. In the mitochondrial

pathway, death stimuli target mitochondria either directly or through transduction by

proapoptotic members of the Bcl-2 family [21]. Cell sensitivity to apoptotic stimuli

depends on the interaction between pro-apoptotic and anti-apoptotic Bcl-2 proteins

[22, 23]. The pro-apoptotic Bcl-2 proteins (Bad, Bax, Bak, etc.) in the cytosol act as

sensors of cellular damage and relocate to the surface of the mitochondria due to

cellular stress [24]. In the mitochondrion, Bax and Bak oligomerization is induced by

p53 which then physically interacts with protective Bcl-xL and Bcl-2 on

mitochondrial surface [25]. The interaction between pro-apoptopic and anti-apoptotic

proteins disrupts the normal function of the anti-apoptotic Bcl-2 proteins, thus

resulting in the formation of pores in the mitochondria through which cytochrome C

and other pro-apoptotic molecules are released [26]. This event eventually leads to the

activation of effector caspases, which are responsible for the cleavage of the key

cellular proteins, that leads to characteristic morphological changes observed in cells

undergoing apoptosis [27].

The cleavage of chromosomal DNA into nucleosomal units is one of the signals of

apoptosis [28]. The caspases play critical role in the process of apoptosis by activating

DNAses, inhibiting DNA repair enzymes and breaking down structural proteins in the

nucleus [29]. As observed from DNA ladder assay, radiation induced fragmentation

of DNA into nucleosomal units in V79 cells caused by an enzyme known as caspase

activated DNase (CAD). This CAD usually exists as an inactive complex with ICAD

(inhibitor of CAD), which is cleaved by caspases (caspase 3) during apoptosis

releasing CAD, that triggers rapid fragmentation of the nuclear DNA [28, 30]. In the

present study, the elevated level of cleaved caspase-3 (active) with radiation treatment


119

correlated the events of DNA damage. PARP, a protein involved in repair of DNA

damage, was found reduced in radiation treated cells, indicated by increased levels of

cleaved PARP by active caspase 3 [31]. The ability of PARP to repair DNA damage

was thus arrested. All these parameters were reversed with PMC-1 pretreatment in

V79 cells exposed to radiation.

3.5. Conclusions

PMC-1 attenuated radiation-induced protein carbonylation, lipid peroxidation

and DNA damage in pBR322. The determined rate constants for the reactions

of PMC-1 with different free radicals [(•OH) and (CCl3O2•)] indicated

antioxidant properties of PMC-1 carried out by pulse radiolysis.

PMC-1 pretreatment decreased the radiation-induced increase in phospho-p53,

p53, Bad, cleaved caspase-3 and cleaved PARP in irradiated V79 cells

compared to radiation control.


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