ameliorative role of quercetin and/or resveratrol on

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92

El-Alfy et al. World Journal of Pharmacy and Pharmaceutical Sciences

AMELIORATIVE ROLE OF QUERCETIN AND/OR RESVERATROL

ON ACROLEIN-INDUCED CLASTOGENESIS IN BONE MARROW

CELLS OF MALE ALBINO MICE MUS MUSCULUS

Nagla Zaky Ibrahim El-Alfy1, Mahmoud Fathy Mahmoudand

2 and Sally Ramadan

Gabr El-Ashry3*

1Professor of Cytogenetics,

2Professor of Cytology and Histology,

3PhD Researcher

Department of Biological and Geological Sciences, Faculty of Education, Ain Shams

University, PO11341, Cairo, Egypt.

ABSTRACT

Acrolein, a highly reactive unsaturated aldehyde, is considered as a

mutagenic environmental pollutant which can cause oxidative stress by

generation of reactive oxygen species. The aim of the present study is

to investigate the cytoprotective role of oral pretreatment with

quercetin (50 mg/ kg body weight) alone, resveratrol (12.5 mg/ kg

body weight) alone and the mixture of both quercetin/ resveratrol

against the clastogenesis of acrolein (10 mg/ kg body weight) in bone

marrow cells of male albino mice Mus musculus by using

chromosomal aberration assay, mitotic index and micronucleus test as

toxicological endpoints. In this study, quercetin and/ or resveratrol

were given to mice for eight days (four days prior to acrolein treatment

followed by other four days along with acrolein treatment). The results

revealed that oral administration of acrolein to mice for four consecutive days significantly (P

< 0.001) increased the incidence of aberrant metaphases, structural and numerical

chromosomal aberrations, micronuclei formation and cytotoxicity in bone marrow cells in

comparison with the control group. Also, pretreatment of mice with quercetin and/ or

resveratrol significantly (P < 0.001) reduced acrolein-induced clastogenesis and cytotoxicity

in the bone marrow cells. No considerable difference was observed between the

cytoprotective effects of quercetin alone, resveratrol alone and also the mixture of both

quercetin/ resveratrol against acrolein-induced clastogenesis and cytotoxicity. However, oral

pretreatment of quercetin alone showed the best protective effect against acrolein-toxicity.

*Corresponding Author

Sally Ramadan Gabr El-

Ashry

PhD Researcher, Department

of Biological and Geological

Sciences, Faculty of

Education, Ain

ShamsUniversity, PO11341,

Cairo, Egypt.

Article Received on

07 March 2021,

Revised on 28 March 2021,

Accepted on 18 April 2021

DOI: 10.20959/wjpps20216-18910

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.632

Volume 10, Issue 5, 92-114 Research Article ISSN 2278 – 4357


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Therefore, natural foods rich in plant polyphenols, particularly quercetin and resveratrol,

should be included in the human daily diet to protect them against the deleterious effects of

clastogenic agents like acrolein.

KEYWORDS: Clastogenesis, Bone marrow cell, Chromosomal aberration, Mitotic index,

Micronucleus test, Mice.

1. INTRODUCTION

Acrolein is a reactive α, ß unsaturated aldehyde which can react with many cellular molecules

including amino acids, proteins and nucleic acids and can arise DNA adducts, DNA damage

and inhibition of DNA repair.[1]

The sources of acrolein that are most relevant to human

exposure are grouped into dietary, endogenous and environmental sources.[2]

It is formed in

cooked foods from carbohydrates, vegetable oils, animal fats and amino acids during heating

of foods and exists naturally in some vegetables, fruits and herbs.[3]

Environmentally, the

ubiquitous presence of acrolein is attributed to incomplete combustion of petroleum fuels,

biodiesel, paper, wood and plastic, smoking of tobacco products and frying of foods in oils.[4]

In addition, acrolein can be generated endogenously by several methods during cellular

metabolism as degradation of threonine by neutrophil derived myeloperoxidase, amine

oxidase mediated catabolism of polyamines such as spermine and spermidine, metabolism of

cancer drugs such as cyclophosphamide and lipid peroxidation of polyunsaturated fatty

acids.[2]

Acrolein interferes with the tissue antioxidant defense system, produces highly

reactive oxygen free radicals[5]

and suppresses the activities of antioxidant enzymes such as

superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT).[6]

The

increased level of acrolein adducts and oxidative stress have been observed in plasma of

patients with cancer[7]

, renal failure[8]

, Alzheimer's disease[9]

and diabetes.[10]

Plant polyphenols are natural antioxidants scavenging free radicals and chelating transition

metal ions.[11]

Moreover, experimental studies have confirmed the selective cytotoxic activity

of polyphenols in cancer cells and the lack of any such action or at least only minimal

cytotoxicity in normal cells.[12,13]

Quercetin (Q) is a polyphenolic flavonoid compound present in large amounts in vegetables,

fruits and tea and it exhibits therapeutic potential including hepatoprotection and the

inhibition of liver fibrosis.[14,15]

The antioxidant activity of quercetin is related to the presence

of a phenolic hydroxyl group in its chemical stucture which is a good hydrogen donor and


94


can react with reactive oxygen and reactive nitrogen species in a termination reaction which

breaks the cycle of generation of new radicals.[16]

During cancer chemotherapy, quercetin is

suggested to play a role in reducing cytogenotoxicity induced by cyclophosphamide by

decreasing oxidative stress and inflammation.[17]

Resveratrol (RES) is a polyphenol and a powerful antioxidant which is known to have anti-

inflammatory, anti-neoplastic, anti-platelet aggregation, anti-fibrotic, anti-allergic and anti-

aging actions and occurs naturally in red grapes, mulberries and peanuts.[18]

The direct

antioxidant effects of resveratrol are probably related to the presence of hydroxyl groups that

can trap reactive oxygen species on the stilbenic skeleton.[19]

No statistically significant

difference was observed in the mean of total chromosomal aberration frequencies in mouse

bone marrow cells of the control group and resveratrol (100 mg/kg) orally administrated

group.[20]

Many reports have observed the anti-mutagenic and anti-carcinogenic activity of

resveratrol by its ability to prevent DNA damage and to increase DNA repair.[21,22]

Accordingly, the present study was designed to evaluate the effect of quercetin and/ or

resveratrol on clastogenesis induced by acrolein.

2. MATERIALS AND METHODS

2.1. Animals

Adult male mice (CD1) of nearly the same age (16-18 weeks old) with an average body

weight 22-26 g (mean 24 ± 2 g) were obtained from the closed colony of Theodor Bilharz

Research Institute, Cairo. All animals were kept under suitable laboratory conditions of

humidity, temperature (25°C ± 2) and light (12 hr light/12 hr dark) and they were fed on

standard rodent pellet diet and supplied with water, during the whole period of

experimentation. All animal procedures in the present study were conducted in accordance

with the ethical guidelines for investigations in laboratory animals and comply with the guide

for the care and use of laboratory animals.[23]

The study also approved by the research ethics

committee of Ain Shams University, Egypt.

2.2. Chemicals

The chemicals used in the present investigation were acrolein (Acr), quercetin (Q) and

resveratrol (Res). They were purchased from Sigma Aldrich (St. Louis, MO, USA). Acrolein

was obtained in the form of clear, colorless liquid, sensitive to light, heat and air. Appropriate

acrolein concentration (10 mg/ kg b. wt.) was freshly prepared directly before treatment by

dilution with distilled water and protected from light and heat. Quercetin (Q) was obtained in


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the form of yellow crystalline powder and its dose (50 mg/ kg b. wt.) was freshly prepared

daily before treatment by suspending it in corn oil and protected from light. Resveratrol was

obtained in the form of off-white to pale yellow powder. Its stock solution was prepared by

dissolving 100 mg of powder in 2 ml of absolute ethanol and storing it in freezing

temperature (at or below 4°C) at dark condition then the required resveratrol concentration

(12.5 mg/ kg b. wt.) was freshly prepared by adding sterile distilled water. The doses were

converted from human dose to mice dose by using multiplication factors for dose conversion

between different species by Paget & Barnes.[24]

2.3. Experimental design

One hundred mice (CD-1) were acclimated for one week and randomly assigned to one of ten

groups, comprising of ten animals each as follows: Group 1: mice served as a control group

and received distilled water (the acrolein’s solvent) orally at same volume as acrolein

treatment. Group 2: mice served as quercetin-vehicle control group and received corn oil

(the quercetin’s vehicle) orally at same volume as quercetin for eight consecutive days.

Group 3: mice served as resveratrol-vehicle control group and received 2% ethyl alcohol

(the resveratrol’s vehicle) orally at same volume as resveratrol for eight consecutive days.

Group 4: mice received oral dose of acrolein (10 mg/ kg) for four consecutive days. Group

5: mice received oral dose of quercetin (50 mg/ kg) for eight consecutive days. Group 6:

mice received oral dose of quercetin (50 mg/ kg) alone for four consecutive days directly

prior to oral treatment with both quercetin (50 mg/ kg) followed by acrolein (10 mg/ kg) for

other four consecutive days. Group 7: mice received oral dose of resveratrol (12.5 mg/ kg)

for eight consecutive days. Group 8: mice received oral pretreatment with resveratrol (12.5

mg/ kg) alone for four consecutive days directly prior to oral treatment with both resveratrol

(12.5 mg/ kg) followed by acrolein (10 mg/ kg) for other four consecutive days. Group 9:

mice received both quercetin (50 mg/ kg, orally) and resveratrol (12.5 mg/ kg, orally) for

eight consecutive days. Group 10: mice received oral pretreatment with quercetin and

resveratrol (50 mg/ kg, 12.5 mg/ kg, respectively) for four consecutive days directly prior to

oral treatment with both quercetin and resveratrol followed by acrolein (10 mg/ kg) for other

four consecutive days. Oral administration of acrolein was separated from regular quercetin

and/or resveratrol treatment by one hour. All the control and treated animals were sacrificed

by cervical dislocation at 24 hr after the last treatment for collection of samples.


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2.4. Chromosomal aberration analysis

Each animal was weighed alive prior to the intraperitoneal injection with colchicine solution

(0.2 ml/ 100 g) and they were sacrificed after two hours. Bone marrow chromosome

preparations were carried out according to the method of Preston et al.[25]

Observation was

made using bright field and most photographs were taken with a 100x oil object. The best

photograph with a spread metaphase stage was chosen for making a karyotype. One hundred

well spread metaphase plates per mouse (500 metaphases for each group) were examined in

the control and all treated groups for both structural and numerical aberration.

2.5. Mitotic index

From the prepared slides, mitotic index (MI) was evaluated by counting the divided cells

among at least 1000 metaphase spreads per each group (five animals per each group) and

expressed in percentage.

2.6. Micronucleus test

In the present study Schmid’s[26]

standard procedure was followed however with slight

modification. Instead of foetal calf serum, 5% bovine albumin (from National Research

Center, Giza, Egypt) was used as suspending medium to collect the bone marrow.[27]

At the

end of experiment, Mice were sacrificed and femurs were trimmed and a blunt needle was

pushed to pierce the marrow cavity. The marrow was flushed through a syringe with 5%

bovine albumin to obtain a fine suspension which was centrifuged at 1000 rpm for 8 to 10

minutes. Then, the supernatant was discarded and fresh suspending medium was added. A

smear was prepared (3-4 slides/animals), slides were air dried overnight, fixed by methanol

for 5 minutes, dried, stained by May-Grunwald and then with a combination of May-

Grunwald and phosphate buffer at pH 6.8 for the proper color differentiation of

polychromatic erythrocytes (PCE’s) and normochromatic erythrocytes (NCE’s). Finally

slides are stained with Geimsa and buffer at pH 6.8 for micronuclei staining. After washing

with distilled water and buffer good slides were dried and mounted. Two thousand PCE’s

were screened per animal and micronucleated PCE’s (MNPCE’s) were recorded.

Consequently, identified normochromatic erythrocytes (NCE’s) were also recorded. Finally,

the percentage of MNPCE and PCE/NCE ratio were calculated for each animal.

2.7. Data analysis

Statistical analyses were carried out using the package software SPSS/PC computer program

(version 16.0). All values were expressed as mean and standard deviation (mean ± SD). The


97


independent samples T-test was used for comparison between means of two groups to

determine if the difference between means is statistically significant or due to sampling error.

A value of (P < 0.05) was considered significant, (P < 0.001) was considered highly

significant and (P > 0.05) was considered insignificant. Chart was drawn using Excel 2010.

3. RESULTS

3.1. Chromosomal aberration analysis

Current results as shown in Table 1 revealed that acrolein (10 mg/ kg) administration for four

consecutive days significantly (p < 0.001) increased the incidence of structural and numerical

chromosomal aberrations in the bone marrow cells of male mice in comparison to the

corresponding control. Table 1 and Figure 1 illustrated that acrolein administration induced

various chromosomal aberrations, which were then further classified into structural

aberrations such as centromeric attenuation (Ca), chromosome gap (Chg), chromatid gap

(Cg), centric fusion (CF), ring chromosome (R), beaded chromosomes (Bch), end to end

association (Ee), pulverized chromosomes (P), sticky chromosomes (S), deletion (D) and

fragment and numerical aberrations in the form of polyploidy. However, the mean of

centromeric attenuation, fragment, deletion and sticky chromosomes were higher as

compared to other chromosomal aberrations. On the other hand, oral pretreatment of plant

polyphenols (quercetin and/or resveratrol) prior to acrolein treatment significantly (p < 0.001)

decreased the mean of total aberrations comparable to that of acrolein-administrated group

(Group 4) which indicated the protective role of quercetin and/ or resveratrol against

clastogenicity of acrolein. In addition, this study observed that pretreatment of quercetin prior

to acrolein treatment gave more effective reduction in the total chromosomal aberrations and

abnormal metaphases than pretreatment of resveratrol or pretreatment of both quercetin/

resveratrol as shown in Figure 2. Quercetin (50 mg/kg) and resveratrol (12.5 mg/kg) were

also not observed to be clastogenic, as there were no significant changes in the frequency of

chromosomal aberrations over the corresponding quercetin-vehicle control group and the

corresponding resveratrol-vehicle control group, respectively.

3.2. Mitotic index

Mitotic index was employed to analyze potential toxicity of acrolein and potential protective

role of quercetin and/ or resveratrol in terms of cell proliferation. Table 2 and Figure 3

revealed that after the oral administration of acrolein dose (10 mg/ kg) for four consecutive

days to mice of group 4, the percentage of mitotic index was highly significant (p < 0.001)


98


decreased compared to the corresponding control group, confirming bone marrow

suppression. Moreover, a highly significant increase in the mitotic index was observed as a

result of pretreatment of quercetin and/or resveratrol prior to acrolein treatment, indicating

the cytoprotective role of them against acrolein-induced bone marrow suppression as shown

in Table 2 and Figure 3. Quercetin (50 mg/kg) and resveratrol (12.5 mg/kg) were also not

observed to be cytotoxic, as the oral pretreatment of each of them alone induced a highly

significant (P < 0.001) increase in the mitotic index over the corresponding quercetin-vehicle

control group and the corresponding resveratrol-vehicle control group, respectively.

3.3. Micronucleus test

The results of micronucleus test were summarized in Table 3. As shown in Figure 4, the

polychromatic erythrocytes were stained light blue to gray and normochromatic erythrocytes

were stained light pink to light yellow. Figure 5 showed that polychromatic erythrocytes with

micronuclei (MNPCEs) are polychromatic erythrocytes have one or more small nuclei (dark

blue in color) as a residual hereditary material remained after erythropoiesis process.

Acrolein induced a significant (P < 0.05) increase in the frequency of micronucleated

polychromatic erythrocytes over the corresponding control. On the other hand, mice treated

with quercetin and/ or resveratrol before and along with acrolein treatment showed a

significant (P < 0.05) reduction in the incidence of micronucleated polychromatic

erythrocytes (MNPCEs) as compared to the acrolein treated group. Oral pretreatment with

quercetin before acrolein administration showed more remarkable decrease in MNPCEs

frequencies than pretreatment with resveratrol alone or with both quercetin/resveratrol.

Moreover, assessment of the ratio of polychromatic erythrocytes to corresponding

normochromatic erythrocytes (PCE/NCE) of control and all treated groups was used as an

index of cytotoxicity and an indicator of the acceleration or inhibition of erythropoiesis. As

shown in Table 3, oral administration of acrolein alone resulted in a highly significant

increase in cytotoxicity of bone marrow cells by decreasing the ratio of PCE/NCE less than

the corresponding control. Also, the anti-cytotoxic potential of quercetin and/ or resveratrol

caused a significant reduction in the cytotoxicity of bone marrow cells by increasing the ratio

of PCE/NCE more than the corresponding acrolein- treated group. Treatment of mice with

quercetin or resveratrol alone without acrolein did not induce any significant variation in the

incidence of micronucleated polychromatic erythrocyte (MNPCE) as compared to the

corresponding quercetin-vehicle control group and the corresponding resveratrol-vehicle


99


control group, respectively. In addition, quercetin and resveratrol were not cytotoxic to the

bone marrow and did not cause a significant decrease in the ratio of PCEs/NCEs.


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Table 1: Average of chromosomal aberration observed in bone marrow cells of male albino mice Mus musculus of the control and all

treated groups (mean±standered deviation).

Group Treatment (mg/ kg)

Mean number (mean±SD) and types of structural and numerical chromosomal

aberrations Total aberrations

(Mean±SD) Ca Chg & Cg CF R Bch Ee P S D F Po

1 Control 12.4±

2.5

8.2±

1.05

6.06±

1.22

10.8±

1.3

1.4±

1.2

7.26±

2.8

2±

0.4

8.3±

1.5

10.1±

4.2

14.1±

.7 0

80.8±

0.220

2 Q-vehicle 17.6±

3.1

9.9±

2.5

6.4±

1.2

12.4±

2.5

0.6±

1.1

9.9±

2.5

1.6±

1

13.1±

0.8

11.2±

3.2

14.5±

1.9 0

97.6±

7.600

3 Res-vehicle 20.2±

2.2

3.4±

1.3

6.6±

1.4

8.4±

2.4

0.8±

1.3

7.2±

2.5

4.5±

0.5

6.7±

1.5

12.1±

2.5

16.1±

1.2

1.8±

0.6

88.1±

0.980

4 Acr (10) 40.4± 2.2

⁎ 17.8± 2.4

⁎ 11.2± 1.2

⁎ 16.9± 0.5

⁎⁎ 6.4± 1.3

⁎ 20.1± 2.2

⁎ 18.3± 1.5

⁎ 22.8± 3.3

⁎ 25.4± 5.06

⁎ 29.9± 2.7

⁎ 13.7± 3.4

⁎ 223.3± 0.940

⁎⁎

5 Q (50) 22.4±

2.5⁎

4.2±

2.8

5±

2.5

9.6±

2.02⁎

4.4±

0.5⁎

10.6±

2.08

7.3±

2.5⁎

13.4±

1.3

7.4±

2.5⁎

9.1±

1.8⁎

1.2±

0.7

94.9±

1.680

6 Q (50) + Acr (10) 32.4± 2.5

⁎ 6.1± 1.2

⁎ 5.4± 1.3

⁎ 11.4± 1.2

⁎ 4.1± 1.8

10± 5

7.5± 0.4

⁎ 19.2± 1.6

⁎ 8.8± 1.3

⁎ 22.5± 2.5

⁎ 2.14±

0.4 129.8± 1.880

⁎⁎

7 Res (12.5) 19.4±

2.6 8.3± 2.8

⁎

7.4± 2.7

8± 2.6

4.9± 2.5

⁎

6.7± 1.5

6.7± 0.8

⁎

12.4± 0.4

⁎⁎

11± 1.2

18.4± 1.4

⁎

0

103.4± 1.740

8 Res (12.5) + Acr

(10)

24±

3.6⁎

11.2±

3.3⁎

9.1±

1.8⁎

14.8±

2.3⁎

2.4±

2.5

13±

2.6⁎

7.6±

1.3⁎

12.4±

2.5⁎

12.4±

6.5

24.1±

6.3⁎

1.8±

0.5⁎

135.1±

1.960⁎⁎

9 Q (50) + Res (12.5) 21±

3.6⁎

7.4±

2.5

3.8±

1.3⁎

15.1±

0.9⁎

2.1±

2

14.6±

9.2⁎

3.2±

1.05⁎

20.3±

5.03⁎

11.8±

1.6

15.2±

4.1

1.4±

1.2

116.2±

1.180

10 Q (50) + Res (12.5)

+ Acr (10) 24.3± 5.1

⁎ 8.8± 3.3

⁎ 6.6± 1.5

⁎ 15.4± 1.7

⁎ 1.6± 1.44

10.7± 2.8

⁎ 4.5± 1.1

⁎ 22.1±

3 19.9± 6.6

⁎ 18.2± 3.7

⁎ 1.9± 0.9

134.5± 0.480

⁎⁎

Five hundred metaphases were scored for chromosomal aberrations per each group (five mice were examined per each group). The significances

were indicated as follow: *Significant (P < 0.05) and **Highly Significant (P < 0.001). Ca, centromeric attenuations; Cg, chromatid gaps; Chg,

chromosome gaps; CF, centric fusions; R, rings; Bch, beaded chromosomes; Ee, end to end association; P, pulverization; S, stickiness; D,

deletion; F, fragments; Po, polyploidy.


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Table 2: Effect of oral pretreatment with quercetin and/ or resveratrol on the score of

divided cells and the percentage of mitotic index (MI %) in bone marrow cells of male

albino mice treated with acrolein (mean±standered deviation).

Group Treatment (Dose mg/ kg)

No. of

examined

mice

No. of

examined

cells/ mice

Score of

divided cells (Mean± SD)

Percentage of

mitotic index (MI %)

(Mean± SD)

1 Control 5 1000 96.2± 4.86826 9.6200± 0.48683

2 Q-vehicle 5 1000 71.6± 8.5322 7.1600± 0.85323

3 Res- vehicle 5 1000 81.8± 4.43847 8.1800± 0.44385

4 Acr (10) 5 1000 53.2±

3.96232⁎⁎

5.3200± 0.39623⁎⁎

5 Q (50) 5 1000 100± 3.93700⁎⁎

10.0000±

0.39370⁎⁎

6 Q (50) + Acr (10) 5 1000 96.2±

4.60435⁎⁎

9.6200± 0.46043⁎⁎

7 Res (12.5) 5 1000 93.6±

4.82701⁎⁎

9.3600± 0.48270⁎⁎

8 Res ( 12.5) + Acr (10) 5 1000 92.4±

3.36155⁎⁎

9.2400± 0.33615⁎⁎

9 Q (50) + Res (12.5) 5 1000 120± 2.00000⁎⁎

12.0000±

0.20000⁎⁎

10 Q (50) + Res (12.5) + Acr

(10) 5 1000

112.8±

13.81304⁎⁎

11.2600±1.38130⁎⁎

The significances were indicated as follow: ⁎

Significant (P < 0.05) and ⁎⁎

Highly Significant

(P < 0.001). MI, mitotic index.

Table 3: The mean and standard deviation of micronucleated polychromatic

erythrocytes (MNPCEs) and PCEs/ NCEs ratio in 6000 polychromatic erythrocytes

(PCEs) and corresponding normochromatic erythrocytes (NCEs) scored in the bone

marrow of three male albino mice Mus musculus of the control group and treated

groups.

Group

Treatment

(Dose mg/

kg)

Total

scored cells/

No. of mice

Total

MNPCEs

Micronuclei

MNPCEs/Total PCEs %

(Mean± SD)

Total

NCEs

Cytotoxicity

PCEs/ NCEs

(Mean± SD)

1 Control 6000/3 47 0.7777± 0.25455 5988 1.0017±

0.00764

2 Q-vehicle 6000/3 119 1.9800± 0.62960

5469 1.0933±

0.01528

3 Res- vehicle 6000/3 143 2.3867± 0.19630 5790 1.0333± 0.

03512

4 Acr (10) 6000/3 373 6.2200± 0.85159⁎

6533 0.9180±

0.00700⁎⁎

5 Q (50) 6000/3 103 1.7200± 0.25534 5570 1.0733±


102


0.02082

6 Q (50) + Acr

(10) 6000/3 147 2.4400± 0.25534

⁎ 5685

1.0500±

0.04000⁎⁎

7 Res (12.5) 6000/3 117 1.9400± 0.19053 5420 1.1033±

0.01155

8 Res (12.5) +

Acr (10) 6000/3 158 2.6367± 0.17214

⁎ 5265

1.1333±

0.00577⁎⁎

9 Q (50) + Res

(12.5) 6000/3 80 1.3300± 0.17000 5920

1.0147±

0.07333

10

Q (50) + Res

(12.5) + Acr

(10)

6000/3 153 2.5533± 0.42253⁎

5930 1.0100±

0.05196⁎

The significances were indicated as follow: ⁎

Significant (P < 0.05) and ⁎⁎

Highly Significant

(P < 0.001). MNPCEs, micronucleated polychromatic erythrocytes; PCEs, polychromatic

erythrocytes; NCEs, normochromatic erythrocytes.

Figure 1: Metaphase chromosomes in mice bone marrow cells. (a) Normal chromosomes

of control group; (b, c and d) Different types of structural chromosomal aberrations in

bone marrow cells of acrolein treated mice of group 4 including Ca, centromeric


103


attenuations; Cg, chromatid gaps; Chg, chromosome gaps; CF, centric fusions; R,

rings; Bch, beaded chromosomes; Ee, end to end association; D, deletion and F,

fragments; (e) P, pulverization which is an extreme fragmentation of chromosomes as a

result of acrolein treatment; (f) Po, polyploidy was the common form of numerical

change which was observed as a result of acrolein treatment. Scale bar, 0.05 µm.

Figure 2: Different types of chromosomal aberrations in mice bone marrow cells

because of pretreatment of quercetin and/or resveratrol prior to acrolein treatment

including Ca, centromeric attenuations; Cg, chromatid gaps; CF, centric fusions; R,

rings; Ee, end to end association; S, stickiness; D, deletion; F, fragments. (a and b) Mice

of group 6 received oral pretreatment of quercetin alone prior to acrolein treatment; (c

and d) Mice of group 8 received oral pretreatment of resveratrol alone prior to acrolein

treatment; (e and f) Mice of group 10 received oral pretreatment of both quercetin and

resveratrol prior to acrolein treatment. Scale bar, 0.05 µm.


104


Figure 3: Chart represents the percentages of mitotic index (MI %) of bone marrow

cells of male albino mice in the control group and all treated groups.

Figure 4: Normal bone marrow smears of male albino mice Mus musculus showing

polychromatic erythrocyte (PCE) and normochromatic erythrocyte (NCE). (a) Control


105


group; (b) Quercetin-vehicle control group; (c) Resveratrol-vehicle control group; (d)

Quercetin-treated group; (e) Resveratrol-treated group; (f) Quercetin/resveratrol-

treated group. Scale bar, 0.05 µm.

Figure 5: Bone marrow smears of male albino mice Mus musculus showing

polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE) and

micronucleated polychromatic erythrocytes (MNPCE). (a, b and c) Acrolein-treated

group; (d) Quercetin/acrolein-treated group; (e) Resveratrol/acrolein-treated group; (f)

Quercetin and resveratrol/acrolein-treated group. Scale bar, 0.05 µm.


106


4. DISCUSSION

The current study investigated the protective effects of the plant polyphenols, quercetin and

resveratrol, against the genotoxicity of acrolein by measuring the frequency of chromosomal

aberrations, mitotic index and micronuclei in bone marrow cells of male albino mice Mus

musculus. Also, the previous study of[28]

investigated the genotoxicity of the widely

prescribed drugs depakine and/or epanutin in mice by using chromosomal aberration assay.

Quercetin and resveratrol were selected because they have been considered representative,

more abundant in human nutrition and more promising in terms of their positive effects in

previous studies.[29,30]

In the present study, chromosomal aberration assay indicated that the

oral administration of acrolein (10 mg/kg) highly significant (P < 0.001) increased the

incidence of structural and numerical chromosomal aberrations in bone marrow cells of male

albino mice. Also, previous in vitro experiments observed that acrolein induced chromosomal

aberrations, sister chromatid exchanges, point mutations and inhibition of DNA repair in

cultured human lymphocytes[31]

and mammalian cells[32]

due to its high binding affinity for

proteins and low molecular weight sulfhydryl compounds such as glutathione and cysteine.

Current study of bone marrow chromosomes revealed that centromeric attenuation, fragment,

deletion and sticky chromosomes occurred more frequently after acrolein administration. The

present finding coincided with the previous study of Saxena et al.[33]

which observed that

acrolein intraperitoneal administration (1 mg/kg/day) to female albino rats for 10 days

induced significant differences in term of chromosome aberrations including chromosome

break (both chromosome and chromatid type), gap and fragmentation.

The data of the present study showed that the oral administration of acrolein (10 mg/kg) for

four consecutive days significantly inhibited mitosis and cell proliferation in bone marrow

tissue of treated male albino mice when compared to that of the control. This finding can be

explained by the ability of acrolein to bind strongly with sulfhydryl groups, reduce proteins

synthesis, disrupt the function of many enzymes, deplete cellular glutathione contents,

decrease G0/G1 phase, decrease nuclear division index and centromere protein and inhibit the

formation of spindle fibers.[34]

Another possible mechanism explaining the cytotoxicity of

acrolein was involved in the study of Horton et al.[35]

which demonstrated that acrolein

inhibited the nuclear factor kappa B (NF-κB) controlling several genes, including those

involved in cell proliferation and apoptosis.


107


Micronucleus test was included in many studies to detect genotoxic effect of many classes of

chemicals in mammalian system.[36-38]

Also, it was reported as the most reliable and widely

used bioassay to assess DNA damage in mammalian cells in vivo, because it could detect

genomic alterations resulting from chromosomal damage and/or damage to the mitotic

apparatus caused by clastogenic agents.[39]

In view of that, this test was used in the present

study to measure the genetic damage in bone marrow cells of treated male albino mice in

comparison with the control group. The current data of the micronucleus test showed a

significant increase (p < 0.05) in the mean of micronucleated polychromatic erythrocytes

(MNPCEs) in mice after the oral administration of acrolein (10 mg/kg) for four consecutive

days when compared to the corresponding control. The present investigation was confirmed

by the data from the previous study of Moghe et al.[40]

which revealed that acrolein induced

micronuclei and DNA damage in human, rats and mouse. In addition, Aydın et al.[41]

demonstrated that the oral administration of acrolein (5 mg/kg/day) six days per week for

30 days increased the frequency of micronuclei and decreased the ratio of polychromatic

erythrocytes (PCEs) in bone marrow of treated rats when compared to control. Also, Habibi

et al.[42]

observed that acrolein could interfere with cellular DNA causing DNA strand breaks,

DNA disintegration, structural and numerical chromosomal aberrations, formation of

micronucleated polychromatic erythrocytes and necrosis. The suppression of immature

erythrocytes (PCE) in relation to mature erythrocytes (NCE) causes decreasing in the ratio of

PCE to NCE which is considered as an important index of cytotoxicity that is routinely

included in micronucleus tests to assess the mutagenicity of chemicals to mammals.[43]

The

results of the present study revealed that the mean of PCE/NCE ratio was significantly (p <

0.001) decreased by acrolein administration (10 mg/kg/day) for four days when compared to

control indicating the cytotoxicity of acrolein in bone marrow tissue. It was reported that

spleen captured and destroyed erythrocytes with micronuclei quickly.[44]

Moreover, previous

study of Ahmed et al.[45]

considered acrolein as an eryptosis stimulating molecule and

indicated its ability to cause loss of erythrocytes, impaired formation of erythrocytes, red

blood corpuscles life span shortening and anemia.

The present investigation demonstrated that neither quercetin nor resveratrol were clastogenic

or cytotoxic at the doses tested. Also, Attia[46]

observed that quercetin at doses equivalent to

50 or 100 mg/kg failed to induce chromosomal aberrations in bone marrow cells of mice

which indicated its non-clastogenicity. The protective effects of quercetin in rodent models of

liver disease were maximal when administered at 50 mg/kg.[47]

In addition, the protective


108


effect of oral pretreatment of resveratrol dose (12.5 mg/kg body weight) was investigated

against the genotoxicity of acrolein according to Mokni et al.[48]

who confirmed that the

optimal protective effect of resveratrol on antioxidant enzyme activities and lipoperoxidation

products was obtained at this dose.

In the current study, oral pretreatment of quercetin was able to protect mice bone marrow

cells against the acrolein-induced clastogenicity by significantly (p < 0.001) decreasing the

mean of total structural and numerical chromosomal aberrations induced by acrolein

administration. This result coincided with Sekeroğlu & Sekeroğlu[49]

who observed a

decrease in the number of chromosomal aberrations induced by an anticancer drug called

methotrexate in bone marrow cells after quercetin administration to mice at a dosage of 50

mg/kg body weight.

Results of the present investigation observed that resveratrol exhibited a cytoprotective role

against the toxicity of acrolein in the bone marrow cells by significantly (p< 0.001)

decreasing the mean of total aberrations comparable to that of alone acrolein administrated

mice. This finding was confirmed previously by various investigators who examined the

antigenotoxic effect of resveratrol. Türkez & Sisman[50]

demonstrated that the high

concentrations of resveratrol were not genotoxic and could minimize the frequency of

chromosome aberrations and sister chromatid exchanges caused by aflatoxin in human

lymphocytes. Türkez & Aydin[51]

observed that the combined application of the most popular

environmental pollutant, Permethrin and the plant derived antioxidant, resveratrol,

significantly reduced the frequency of chromosomal aberrations and the formation of sister

chromatid exchanges in cultured human lymphocytes in comparison with alone Permethrin-

treated cultures.

The present data of micronucleus assay revealed that the antigenotoxic potential of the plant

polyphenols (quercetin and/or resveratrol) protected bone marrow tissue of mice against

genotoxicity induced by acrolein by significantly reducing the mean of (MNPCEs) and

significantly increasing the ratio of (PCE/NCE) when compared to acrolein-treated group.

Previously, quercetin was observed to be effective in decreasing the micronuclei frequency

and protecting cultured rat peripheral lymphocytes against nicotine-induced cellular and

DNA damage.[52]

Importantly, quercetin was able to enter erythrocytes to prevent the

oxidative damage induced by acrolein in erythrocytes because of glutathione depletion which

was followed by the release of free iron, lipid peroxidation and consequent hemolysis.[53]

The


109


role of resveratrol to prevent chromosomal abnormalities and reduce micronuclei formation

was explained by its ability to restore the levels of intracellular antioxidants such as

glutathione peroxidase, superoxide dismutase and catalase activity.[54]

Also, the stilbenic

structure of resveratrol was responsible for its strong antioxidant activity due to the presence

of a hydroxyl group which could trap reactive oxygen species.[55]

5. CONCLUSION

The findings of the present investigation demonstrated that acrolein, a toxic

cyclophosphamide metabolite and a major component in the gas phase of cigarette smoking,

automobile exhaust, over-heated oils, fried foods and forest fires, was highly clastogenic and

cytotoxic as it induced a very harmful genetic damage in the examined bone marrow cells of

male albino mice Mus musculus. Therefore, acrolein exposure should be limited by regulating

its levels in foods and in tobacco products or preventing its huge emissions into environment.

In addition, the observed protective effects of quercetin and resveratrol indicated that they

can be used as nutritional supplements or as a part of functional foods for geno protective

treatment against acrolein induced genotoxicity in vivo.

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