Comparison of the prevention of aflatoxin B1-induced genotoxicity by quercetin and quercetin pentaacetate

Article abstract of DOI:10.1016/S0960-894X(02)00478-X

Earlier work carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins in preventing the genotoxicity of aflatoxin B₁ (AFB₁). We have in this report extended the observations to quercetin pentaacetate

Full text of DOI:10.1016/S0960-894X(02)00478-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2579–2582
Comparison of the Prevention of Aflatoxin B1-Induced
Genotoxicity by Quercetin and Quercetin Pentaacetate
Ekta Kohli,a Hanumantharao G. Raj,a Ranju Kumari,a Vishwajit Rohil,a
Narendra K. Kaushik,b Ashok K. Prasadb and Virinder S. Parmarb,*
aDepartment of Biochemistry, V. P. Chest Institute, University of Delhi, Delhi-110 007, India
bBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007, India
Received 22 October 2001; accepted 20 June 2002
Abstract—Earlier work carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins in preventing
the genotoxicity of aflatoxin B1 (AFB1). We have in this report extended the observations to quercetin pentaacetate (QPA), which
unlike quercetin (Q) has demonstrated time-dependent inhibition of liver microsome catalysed AFB1 epoxidation as measured
by AFB1 binding to DNA. The action of QPA is similar to that of the acetoxy 4-methylcoumarins in that they are acted upon
by microsomal transacetylase leading to modulation of catalytic activities of certain enzymes (such as P-450 enzymes, NADPH
cytochrome C reductase and glutathione S-transferase) possibly by way of protein acetylation. In the present work, we have
documented the transacetylase-mediated action of QPA in preventing genotoxicity due to AFB1. # 2002 Elsevier Science Ltd. All
rights reserved.
Introduction
marin (DHMC), the deacetylated product of DAMC,
failed to elicit the said effects. These results prompted us
to postulate the existence of an enzyme in microsomes
that can possibly catalyse the transfer of acetyl groups
of DAMC to P-450 apoprotein and thereby cause inhib-
ition of microsome catalysed AFB1–DNA binding both
in vivo and in vitro.7,11 Work carried out by us revealed
the action of transacetylase on several acetylated xeno-
biotics leading to the conclusion that such a transacetyl-
ase in general can be termed as acetoxy drug: protein
transacetylase.12 In the present work, we have provided
evidences for the action of transacetylase on a flavone
acetate, namely quercetin pentaacetate (QPA) and the
implications on P-450 catalysed reaction, such as AFB1
epoxidation and genotoxicity.
Several polyphenolic compounds have demonstrated
antimutagenic and anticarcinogenic activities in cell
cultures as well as animal models.1,2 Among poly-
phenolic compounds, flavonoids have come under ser-
ious scrutiny regarding their ability to prevent chemical
and radiation induced genotoxicity. Flavonoids in gen-
eral inhibit cytochrome P-450-mediated formation of
reactive intermediates of several classes of chemical
carcinogens and mutagens which damage cellular DNA
by way of adduct formation.3À5 Accordingly, quercetin
has been shown to inhibit P-450 catalysed activation of
several chemical carcinogens including aflatoxin B1
(AFB1).6 Our laboratory has carried out an in-depth study
of another class of polyphenolics, namely hydroxy
4-methylcoumarins and their acetyl derivatives on the
microsome catalysed epoxidation of AFB1 and related
biological effects.7À10 The results of these investigations
revealed the unique action of the model compound
7,8-diacetoxy-4-methylcoumarin (DAMC) to cause a dra-
matic irreversible inhibition of microsome-mediated
AFB1–DNA binding, while 7,8-dihydroxy-4-methylcou-
Materials and Methods
Chemicals
[3H] AFB1-G was obtained from Moravek Biochemicals
(Brea, CA, USA). Quercetin(Q, 1), NADPH, calf thy-
mus DNA, cytochrome C and AFB1 were purchased
from Sigma Chemical Co, St. Louis, MO (USA). Quer-
cetin was acetylated by the acetic anhydride/pyridine/
DMAP method to obtain quercetin pentaacetate (QPA,
*Corresponding author. Tel.: +91-11-766-6555; fax: +91-11-766-
7206; e-mail: virparmar@yahoo.co.in
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00478-X

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E. Kohli et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2579–2582
2), which was fully characterised from its spectral data.
7,8-Dihydroxy-4-methylcoumarin (DHMC, 3) and its
diacetyl derivative (DAMC, 4) were obtained by our
earlier published procedure.7
AFB1-induced micronuclei formation in bone marrow
cells
Treatment. Male albino rats of wistar strain (150–200 g)
were administered, separately, the test compound (300
mg/kg b wt dissolved in 0.1 mL DMSO) intraperi-
toneally (ip), followed by a second dose of the test
compound along with a dose of AFB1 (4 mg/kg b wt in
DMSO). A group of rats were injected with AFB1
alone, while control animals received DMSO alone. The
animals were sacrificed 26 h after the last injection.
Isolation of bone marrow, preparation of smear and
staining
Bone marrow cells were isolated by flushing femora of
rats with 2 mL of Hanks’ Balanced Salt Solution (HBSS).
For preparation of smear and staining, the method of
Schimd16 was followed as adapted by Raj et al.17
Assay of liver microsome catalysed activation of NADPH–
cytochrome C reductase by quercetin and quercetin
pentaacetate
The method consisted of preincubation of the test com-
pound with microsomes, followed by the addition of
substrates for the reductase assay (cytochrome C and
NADPH) as described earlier.8 Rat liver microsomes
(25 mg protein) were separately mixed with a fixed con-
centration of the test compound (DAMC/DHMC/
QPA/Q added in 50 mL of DMSO), phosphate buffer
(0.05 M, pH 7.7) and water to make up to 0.8 mL. The
contents (scaled up as per requirement) were pre-
incubated at 37 ꢀC in a shaking water bath. The aliquots
(0.5 mL portion) were removed periodically into the
spectrophotometer cuvette (1 cm light path) containing
0.1 mM EDTA, 36 mM cytochrome C and 1 mM
NADPH in a total volume of 1 mL, the progress of the
reaction was monitored at 550 nm.
Animals
Male albino rats of wistar strain weighing around 150–
200 g, fed on rat chow supplied by Hindustan Lever
Ltd., Mumbai (India) were used.
Preparation of liver microsomes
Male rats were sacrificed, liver excised and microsomes
prepared as described earlier.13
Effect of test compounds on liver microsome catalysed
AFB1–DNA binding
Rat liver microsomes (1 mg protein) were mixed sepa-
rately with the test compound (DAMC/DHMC/QPA/
Q, 100 mM) and phosphate buffer (100 mM, pH 7.4)
and water to make the volume 0.8 mL. The contents
(scaled up as per requirement) were preincubated at
37 ꢀC in a shaking water bath. The aliquots (0.5 mL
portion) were removed periodically into another set of
tubes containing [3H]AFB1 (250 mCi/mM) in 20 mL
DMSO, 0.1 mg calf thymus DNA, 2 mM NADPH and
made to a final volume of 1 mL and incubated for 30
min at 37 ꢀC. At the end of incubation, 2 mL of the
extraction mixture (phenol/chloroform/isoamyl alcohol
50:50:1 v/v/v) and 0.9 mg calf thymus DNA as the car-
rier were added. The DNA was isolated by the proce-
dure of Wang and Cerutti14 and the recovery of DNA
was 50–70%. Isolated DNA was dissolved in 0.1 M
NaCl, one aliquot (0.2 mL) was added to a vial con-
taining 8 mL of Bray’s scintillation fluid for determina-
Table 1. Influence of quercetin (Q) and quercetin pentaacetate (QPA)
on liver microsome catalyzed AFB1–DNA binding in vitro: compar-
ison with hydroxy- and acetoxy-4-methylcoumarins
Test compd
QPA
Pre-incubation
time (min)
AFB1–DNA binding
(% inhibition)
10
20
30
6.70
12.80
18.00
Q
10
20
30
5.70
5.20
6.20
DAMC
DHMC
10
20
30
19.00
37.50
56.80
tion of radioactivity using
a Liquid Scintillation
Counter (Beckman Model LS 6000).15 Results of [3H]
AFB1 binding to DNA were corrected for DNA
recovery and expressed as [3H] AFB1 bound/
mgDNA/30 min. In this assay procedure, samples
with DMSO substituting for the test compound
served as the control.
10
20
30
8.50
8.20
8.90
The concentration of the test compound included in the incubation
was 100 mM. The values are an average of three observations with
variation <5%. The details are given under Materials and Methods.

E. Kohli et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2579–2582
2581
Table 2. Effect of quercetin (Q) and quercetin pentaacetate (QPA) on
AFB1-induced micronuclei formation in rat bone marrow cells
micronuclei incidence in bone marrow cells (Table 2).
DHMC was found ineffective in significantly reducing
the micronuclei induction in bone marrow cells by
AFB1 (results not shown here) although Q proved a lit-
tle better (Table 2). The role of tranacetylase in the
action of QPA and DAMC is further confirmed by the
results described in Table 3. We have established in our
previous publications8 that DAMC through the action
of TAase drastically activates the catalytic activity of
NADPH cytochrome C reductase. Q, like DHMC is
ineffective in causing the time dependent activation of
the reductase, while QPA is effective in producing irre-
versible activation of the reductase (Table 3). Several
reports have cited the potential of Q to inhibit P-450-
linked MFO activities, such as epoxidation of AFB1 and
benzo(a)pyrene.5,6 Our results (Table 2) denote an
important observation that QPA, like DAMC is an
irreversible inhibitor of AFB1-epoxidation catalyzed by
liver microsomal P-450. We have earlier reported acet-
ylation of recombinant glutathione S-transferase iso-
form 3-3 by DAMC catalyzed by hepatic microsomal
transacetylase.18 Accordingly our earlier observation
that the P-450 activities were inhibited by DAMC could
possibly be due to the acetylation of P-450 apoprotein.19
These results explain that QPA and DAMC are superior
to their hydroxy analogues in preventing the induction
of genotoxicity due to AFB1 (Table 2).These observa-
tions indicate that the action of Q and QPA is not only
confined to the inhibition of P-450 activities, but they
also modulate many other cellular activities.20,21 These
results further confirm our hypothesis that acetylated
polyphenols (DAMC, QPA, etc.) are acted upon by the
microsome membrane bound transacetylase resulting in
the possible acetyl CoA-independent acetylation of
enzyme proteins with altered catalytic activity as high-
lighted in this report.
Group Group description N
Micronucleated cells/1000 cells
Mean
95% Confidence limits
1
2
3
4
5
6
7
8
Control
AFB1alone
Q
4
4
4
4
4
4
4
4
1.75
7.25
2.00
2.25
6.00 (22.72%)
4.75 (45.45%)
2.25
0.6666–2.8334
6.6842–7.8158
1.0760–2.9240
1.6842–2.8158
5.0760–6.9239
4.1842–5.3158
1.6842–2.8158
2.8118–4.6882
QPA
Q+AFB1
QPA+AFB1
DAMC
DAMC+AFB1
3.75 (63.63%)
N, Number of animals in each group; P<0.001: (Group 2 vs Group 1;
Group 3 vs Group 2; Group 4 vs Group 2; Group 6 vs Group 2;
Group 6 vs Group 4; Group 5 vs Group 3; Group 5 vs Group 1;
Group 8 vs Group 2; Group 7 vs Group 2); P<0.005: (Group 6 vs
Group 1); P<0.05: (Group 5 vs Group 2; Group 6 vs Group 5; Group
8 vs Group 7; Group 8 vs Group 1; Group 8 vs Group 5); NS (not
significant): (Group 5 vs Group 1; Group 4 vs Group 1; Group 4 vs
Group 3; Group 7 vs Group 1; Group 8 vs Group 6).
Results and Discussion
The relative activities of Q and QPA to prevent
AFB1-induced genotoxicity are reported here. The pre-
incubation of rat liver microsomes with QPA resulted in
the time-dependent inhibition of AFB1 binding to calf
thymus DNA in vitro (Table 1). It is evident from the
results (Table 1) that the polyphenols (Q and DHMC)
failed to produce time-dependent inhibition of liver
microsome catalyzed AFB1-epoxidation (measured as
AFB1–DNA binding) unlike their corresponding acet-
oxy derivatives. The results documented in Table 2
highlight a comparative account of Q and QPA to pre-
vent the clastogenic action of AFB1 in bone marrow
cells. The pretreatment of rats with QPA and DAMC
caused considerably higher reduction of AFB1-induced
Table 3. Influence of quercetin pentaacetate (QPA) and quercetin (Q)
by transacetylase catalyzed time dependent activation of NADPH
cytochrome C reductase
Acknowledgements
We thank the Danish International Development
Agency (DANIDA) and the Council for Scientific and
Industrial Research (CSIR, New Delhi) for financial
assistance.
Test compd
Preincubation
time (min)
NADPH cytochrome
C reductase
(% activation)
QPA (5 mM)
5
10
20
30
7.10
13.30
25.00
37.00
References and Notes
Q (5 mM)
5
10
20
30
3.70
3.30
5.40
5.70
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