Effect of Sapthaparna (Alstonia scholaris Linn) in modulatingthe benzo(a)pyrene-induced forestomach carcinogenesis in

mice

Ganesh Chandra Jagetia *, Manjeshwar Shrinath Baliga, Ponemone Venkatesh

Department of Radiobiology, Kasturba Medical College, Manipal 576119, Karnataka, India

Received 16 January 2003; received in revised form 2 April 2003; accepted 4 April 2003

Abstract

The chemopreventive effect of various doses of hydroalcoholic extract of Alstonia scholaris (ASE) was studied on the

benzo(a )pyrene (BaP) induced forestomach carcinoma in female mice. The treatment of mice with different doses, i.e. 1,

2 and 4 mg/ml ASE in drinking water before, during and after the treatment with carcinogen, exhibited

chemopreventive activity. The highest activity was observed for 4 mg/ml ASE, where the tumor incidence (93.33%)

was reduced by 6.67%. Similarly, the tumor multiplicity reduced (61.29%) significantly (P B/0.02) at 4 mg/ml in the

pre�/post-ASE treated group. However, the pre or post-treatment of mice with 4 mg/ml ASE did not show

chemopreventive activity. These findings are corroborated by micronucleus assay, where treatment of mice with ASE

before, during and after carcinogen treatment reduced the frequency of micronuclei (MN) in the splenocytes in a dose

dependent manner. The MN frequency reached a nadir at 4 mg/ml ASE, the highest drug dose which showed maximum

chemopreventive action. The ASE treatment not only reduced the frequency of splenocytes bearing one MN but also

cells bearing multiple MN indicating the efficacy of ASE in inhibiting mutagenic changes induced by BaP. The pre or

post-treatment of mice with 4 mg/ml ASE also significantly reduced the frequency of BaP-induced MN in the

splenocytes of treated animals.

# 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Alstonia scholaris ; Carcinogenesis; Benzo(a )pyrene; Forestomach papillomas; Splenocytes; Micronuclei

1. Introduction

Environment pollutants are one of the main risk

factors for the induction of cancer that is con-

sidered as a major public health concern and

leading cause of death in both developing and

developed countries. These pollutants include

benzo(a )pyrene (BaP), which is a polycyclic aro-

matic hydrocarbon (PAH) formed by the pyrolytic

process during smoking of cigarettes and other

tobacco products as well as in combusted organic

matter in automobile exhaust. BaP is frequently

used as a representative indicator of total PAH

levels. It is a complete carcinogen, as it produces

* Corresponding author. Tel.: �/91-8252-57-1201x22814;

fax: �/91-8252-57-0062/1927.

E-mail address: gc.jagetia@kmc.manipal.edu (G.C. Jagetia).

Toxicology Letters 144 (2003) 183�/193

www.elsevier.com/locate/toxlet

0378-4274/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/S0378-4274(03)00205-4

initiation and promotion of carcinogenesis (Halli-

well and Gutteridge, 1989).

Cancer chemoprevention is a mean of cancer

control by pharmacological intervention of the

occurrence of the disease using chemical com-

pounds. Recent events suggest that new emphasis

in the development of medical treatment of human

disease will be intimately connected to natural

products. The use of medicinal plants in modern

medicine for the prevention or treatment of cancer

is an important aspect. For this reason, it is

important to identify anti-tumor-promoting

agents present in medicinal plants commonly

used by the human population, which can inhibit

the progression of tumor.

Alstonia scholaris a tree belonging to the family

Apocynaceae, has been used since time immemor-

ial in the folklore and traditional systems of

medicine in India, to treat several diseases. The

ripe fruits of the plant are used in syphilis insanity

and epilepsy. It is also used as a tonic, antiperiodic

and anthelmintic. The milky juice of A. scholaris

has been applied to treat ulcers. The bark is the

most extensively used part of the plant and is used

in many compound herbal formulations (Nad-

karni, 1976). It is a bitter tonic, alternative and

febrifuge and is reported to be useful in the

treatment of malaria, diarrhea and dysentery

(Satyavati et al., 1987; Warrier et al., 1996;

CHEMEXCIL, 1992; Nadkarni, 1976). A. scho-

laris has also been reported to inhibit liver injuries

induced by carbon tetrachloride, beta-D-galacto-

samine, acetaminophen and ethanol (Lin et al.,

1996). The chief constituents of the bark of A.

scholaris are the alkaloids alstonidine, alstonine,

alstovenine, chlorogenic acid, chlorogenine, ditain,

ditaine, ditamine, echicaoutchin, echicerin, echir-

etin, echitamine, echitein, echitenin, echitin, por-

phyrine, porphyrosine, reserpine, venenatine,

villalstonine pleiocarpamine, O -methylmacralsto-

nine macralstonine O -acetylmacralstonine, villal-

stonine, macrocarpamine, corialstonine and

corialstonidine, and the triterpenoids lupeol linole-

ate, lupeol palmitate and alpha-amyrin linoleate

(Rastogi and Mehrotra, 1990; Gandhi and Vi-

nayak, 1990; Keawpradub et al., 1997, 1999; Rajic

et al., 2000).

As far as the authors are aware, the chemopre-ventive activity of A. scholaris has not been

eva1uated. Therefore, the present study was un-

dertaken to evaluate the chemopreventive activity

of A. scholaris in the mice against the BaP-induced

forestomach carcinoma in mice.

2. Materials and methods

2.1. Preparation of the extract

The identification of the plant A. scholaris R.

Br. (family Apocynaceae) was done by Dr G.K.

Bhat (a well known taxonomist of this area)

Department of Botany, Poorna Prajna College,

Udupi, India and the herbarium specimen hasbeen stored with us. The non-infected stem bark of

the tree was carefully peeled off, shade dried, and

coarsely powdered with the help of a hand club.

The plant material was then extracted with n -

hexane in a Soxhlet apparatus at 55 8C for ten

cycles and dried at 40 8C overnight. The hexane

free plant material was further extracted with 85%

ethyl alcohol in a Soxhlet apparatus extensively for3 days. The cooled liquid extract was concentrated

by evaporating its liquid contents, with an approx-

imate yield of 18%. Henceforth, the extract of A.

scholaris will be called as ASE.

2.2. Animal care and handling

The animal care and handling were done

according to the guidelines set by the WorldHealth Organization, Geneva, Switzerland and

the INSA (Indian National Science Academy,

New Delhi, India). Eight to ten-week-old female

Swiss albino mice weighing 24�/26 g were selected

from an inbred colony maintained under the

controlled conditions of temperature (239/2 8C),

humidity (509/5%) and light (14 and 10 h of light

and dark, respectively). The animals had freeaccess to the sterile water and food (cracked wheat

50%, bengal gram 40%, milk powder 4%, yeast

powder 4%, sesame oil 0.75%, cod liver oil 0.25%,

salt 1%). Four animals were housed in a poly-

propylene cage containing sterile paddy husk

(procured locally) as bedding throughout the

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193184

experiment. The study was approved by theInstitutional Animal Ethical Committee.

2.3. Induction of BaP-induced forestomach

tumorigenesis

Benzo(a)pyrene [BaP, purity �/98% Cat no. B

1760] was procured from Sigma Chemical Com-

pany, St. Louis, MO, USA. The BaP-induced

forestomach tumorigenesis in mice was performedaccording to Wattenberg et al. (1980) with minor

modifications (Nagabhushan and Bhide, 1987).

The animals were administered with 100 ml sesame

oil or 1 mg of BaP in 100 ml sesame oil by p.o.

gavage twice a week for 4 weeks (a total of eight

administrations). The animals were sacrificed 14

weeks after the last administration of BaP.

2.4. Preparation of drug and mode of

administration

The drug was dissolved in double distilled water

(DDW) and filter sterilized. The sterile drug was

added to cooled autoclaved drinking water. The

drug/drinking water was replaced with fresh drug/

drinking water daily (every 24 h) in amber colored

bottles and the leftover was discarded.

2.5. Experimental

The female Swiss albino mice of 4�/5-week-old

were divided into the following groups.

2.5.1. STW alone

The animals of this group received sterile tap

water (STW) as drinking source throughout the

study period.

2.5.2. SMO alone

The animals of this group were injected with 100

ml of sesame oil (SMO) by oral gavage twice a

week for 4 consecutive weeks.

2.5.3. BaP alone

This group of animals was administered 1 mg of

BaP in 100 ml sesame oil by oral gavage twice a

week for 4 weeks.

2.5.4. ASE (pre�/post treatment)

The animals of this group received 1, 2 and 4

mg/ml of ASE as sole source of drinking water for

5 days a week, for 2 weeks before treatment with

BaP followed by the concomitant treatment with

ASE and BaP or STW for 4 weeks during and 2

weeks after the last dose of BaP or sesame oil.

Once the treatment was over, the mice were

provided with normal sterile drinking water forrest of the study period.

2.5.5. ASE (pre-treatment)

The animals of this group received 4 mg/ml of

ASE as sole source of drinking water for 5 days a

week, for 2 weeks. Forty-eight hours before the

first administration of BaP or sesame oil the ASEsource was withdrawn and the mice were provided

with normal drinking water for rest of the study

period.

2.5.6. ASE (post-treatment)

The animals of this group were provided with

normal drinking water during the carcinogen

treatment for 4 weeks. Forty-eight hours afterthe last administration of BaP or sesame oil, the

animals were administered 4 mg/ml ASE for 8

weeks as described above.

The body weights of the animals from each

group were recorded at the beginning of the

experiment and at the termination of the experi-

ment. The mice were sacrificed 14 weeks after the

last dose of BaP. Ten percent phosphate bufferedformalin was immediately injected into the sto-

mach, so that it would be distended and fixed. The

forestomach was cut open longitudinally and kept

in 10% buffered formalin for 24 h for fixation. The

stomach papillomas that were 1 mm or larger in

diameter were counted under a stereozoom micro-

scope (Leica Microsystems, GmbH, Wetzlar, Ger-

many). Formalin fixed forestomach was embeddedin paraffin wax and processed for histology. The

hematoxylin and eosin stained slides were scored

in blind fashion.

The chemopreventive tumor response was as-

sessed on the basis of tumor incidence, mean and

multiplicity of tumors as follows:

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193 185

Tumor incidence

�Number of animals having tumors

Tumor Multiplicity

�Total number of tumors scored

Total number of animals with tumors

The tumor incidence and multiplicity were

compared between the animals fed with the BaP

and the ASE treatment groups. The statistical

significance in the alteration in the body weightsafter various treatments was determined using

Student’s t-test. The tumor incidence and multi-

plicity are expressed as the percentage of animals

with tumors and the analysis was carried out using

x2-test.

2.6. Splenocyte culture

The splenocytes were cultured as described by

Jagetia et al. (2001). Briefly, five animals from

each group were killed by cervical dislocation at

the end of the experiment and thoroughly wiped

with 70% ethanol and their abdominal cavity

opened with the help of sterile scissors and forceps

in an aseptic environment. The spleens of theanimals were removed aseptically and washed in

the sterile phosphate buffered saline (PBS). The

splenocytes were isolated and cultured in RPMI-

1640 medium containing 10% fetal calf serum

(FCS) and concanavalin-A as mitogen. The sple-

nocytes were incubated at 37 8C and allowed to

grow for 72 h. Duplicate cultures were set up for

each animal for each drug dose.The micronuclei (MN) were prepared according

to the method of Fenech and Morley (1985) with

minor modifications. Briefly, 40 h after the initia-

tion of the splenocyte culture, 3 mg/ml of cytocha-

lasin-B was added to the cultures. The cells were

allowed to grow for another 32 h. The splenocytes

were centrifuged, subjected to mild hypotonic

treatment (0.7% ammonium oxalate) so as to

retain the cytoplasm and fixed in Carnoy’s fixative

(3:1 methanol, acetic acid). The cells were centri-

fuged again and resuspended in a small volume offixative and dropped on to precleaned coded slides

to avoid observer’s bias.

Cells were stained with 0.125% acridine orange

(BDH, England, Gurr Cat. No. 34001 9704640E)

in Sorensen’s buffer (pH 6.8). The slides were

washed twice in Sorensen’s buffer and observed

under a fluorescent microscope, equipped with

450�/490 nm BP filter set with excitation at 453 nm(Carl Zeiss Photomicroscope III, Germany), using

a 40�/ Neofluar objective. A minimum of 2000

(1000 per culture) binucleate cells (BNC) with

well-preserved cytoplasm was scored from each

animal for each drug dose for the presence of MN

and the frequency of micronucleated binucleate

splenocytes (MNBNC) was determined. A mini-

mum of four animals was used for each drug dose.The MN identification was done according to the

criteria of Countryman and Heddle (1976) and

Krisch-Volders et al. (2000). The statistical sig-

nificance was determined using one way analysis

of variance (ANOVA) with the application of

Bonferroni’s Post-hoc test.

3. Results

3.1. Forestomach carcinogenesis

Female Swiss albino mice were administeredBaP by oral gavage, twice a week for 4 weeks (a

total dose of 8 mg of the carcinogen), so as to

ensure a high incidence as well as high yield of

forestomach tumors per animal. Simultaneously,

the animals were provided with either sterile

drinking water or ASE dissolved in drinking water

Mean�Lowest number of tumors � Highest number of tumors

2

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193186

to determine their ability to counteract the actionof the carcinogen.

The chemopreventive effect of ASE on the BaP-

induced forestomach papillomas in mice is pre-

sented in Table 1. The administration of 1.0 mg

BaP by oral gavage twice a week for 4 weeks

resulted in 100% incidence of forestomach tumors

with a mean of seven tumors per mouse, which

were histologically considered to be papillomas, asagainst no tumors seen in the SMO, ASE and

STW treated control (without BaP) animals.

The treatment of mice with 1, 2 and 4 mg/ml

ASE as sole source of drinking water for 2 weeks

before, during and 2 weeks after the last dose of

BaP in the pre�/post treatment group brought

about 21.43, 28.57 and 50% reduction in the tumor

multiplicity (Table 1). The reduction in the tumorincidence was observed only in the 4 mg group,

where a reduction of 6.67% was observed.

When 4 mg/ml ASE was given to mice as sole

source of drinking water during the post-initiation

period, starting at 48 h after the last dose of BaP

(post-treatment) and continued for 8 weeks the

tumor multiplicity incidence reduced significantly

(91.93%) when compared with the BaP treated(100%) animals (Table 2). However, the tumor

incidence remained unaltered (100%). The overall

tumor multiplicity was higher in the animals that

were treated with 4 mg/ml of ASE during the post-

treatment (92.85%) than in the pre�/post treatment

group (50%) and was 1.86-fold greater whencompared with the pre�/post treatment group.

Pretreatment of mice with 4 mg/ml of ASE did

not significantly alter the tumor multiplicity and

tumor incidence when compared with the non-

drug treated control.

3.2. Micronucleus assay

The treatment of mice with oil or various doses

of ASE did not significantly alter the frequency of

MNBNC, while treatment of mice with 8 mg of

BaP for the induction of forestomach carcinoma

resulted in 4.75-fold elevation in the frequency of

MNBNC, when compared with spontaneous fre-

quencies (Table 3). The animals that were treated

with different doses of ASE before and after thetreatment of carcinogen exhibited a dose depen-

dent decline in the frequency of MNBNC when

compared with the BaP treated animals, and the

lowest frequency was observed for 4 mg/ml ASE

(Table 3). The treatment of mice with BaP also

induced cells bearing two and three MN, which

were significantly higher than the oil treated

group, while the treatment of mice with differentdoses of ASE before and after BaP treatment

significantly reduced the frequency of MNBNCs

with two and three MN when compared with BaP

alone (Table 3). This reduction was 2 and 6.5-folds

for MNBNC with two and three MN, respectively,

Table 1

Chemopreventive effect of various doses of ASE in the BaP-induced forestomach carcinogenesis in mice

Drug dose (mg/ml) Carcinogen (8 mg) Tumor incidence Mean number Tumor multiplicity Mean body weight (g)

Initial weight Final weight

ASE 0 pre�/post DDW 0 (0/5) �/ �/ 22.119/0.85 32.129/0.92

ASE 0 pre�/post Oil only 0 (0/5) �/ �/ 21.949/0.92 32.259/0.87

ASE 1 pre�/post Oil only 0 (0/5) �/ �/ 22.369/0.69 33.369/0.84

ASE 2 pre�/post Oil only 0 (0/5) �/ �/ 22.819/0.86 33.789/0.68

ASE 4 pre�/post Oil only 0 (0/5) �/ �/ 22.679/0.84 34.219/0.98

ASE 0 pre�/post Oil�/BaP 100 (20/20) 7.0 (100) 6.2 (100) 22.459/0.56 28.869/1.36

ASE 1 pre�/post Oil�/BaP 100 (15/15) 5.5 (78.57)d 5.6 (90.32)d 22.329/0.75 29.489/1.18a

ASE 2 pre�/post Oil�/BaP 100 (15/15) 5.0 (71.43)d 5.2 (83.87)d 22.299/0.68 30.239/1.21b

ASE 4 pre�/post Oil�/BaP 93.33 (14/15)d 3.5 (50)b 3.8 (61.29)b 22.189/0.83 31.759/1.11c

BaP, Benzo(a )pyrene; ASE, A. scholaris extract; a, P B/0.01; b, P B/0.02; c, P B/0.001; d, non significant (comparisons done with

BaP treated group). Number in parentheses in column 3 indicates number of animals with tumor/total number of animals used, while

in column 4 and 5 denotes percent decrease in the occurrence of tumors when compared with the concurrent BaP alone group.

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193 187

after 4 mg/ml ASE pre- and post-treatment. The

decline in MNBNC with two and three MN was

dose dependent. The higher was the dose the

greater was the decline.

The frequency of MNBNC exhibited a signifi-cant decline in the mice treated with 4 mg/ml ASE

either before or after BaP treatment. The frequen-

cies of MNBNC bearing two MN was significantly

lower in the animals treated with the 4 mg/ml ASE

either before or after BaP administration (Table

3). This decline in the MNBNC bearing two MN

was approximately 2-folds in the post-treatment

group, when compared with the BaP alone treatedgroup (Table 3).

4. Discussion

The most promising approach to reduce the

number of cancer cases worldwide may be to

inhibit the occurrence of cancer by pharmacologi-

cal intervention, i.e. chemoprevention. This can be

achieved by the use of certain chemicals or non-nutrient dietary constituents. Chemoprevention,

therefore, is the means of cancer control in which

the occurrence of disease, as a consequence of

exposure to carcinogens, can be slowed, blocked,

or reversed by the administration of one or more

naturally occurring or synthetic compounds (Wat-

tenberg, 1990; Kelloff et al., 1994; Morse and

Stoner, 1993; Stoner and Mukhtar, 1995). Che-

moprevention also deals with the chemotherapy of

precancerous lesions, which are called preinvasive

neoplasia, dysplasia, or intraepithelial neoplasia,

depending on the organ system (Kelloff et al.,

1994). Chemopreventive agents can be targeted by

intervention at the initiation, promotion, or pro-

gression stage of multistage carcinogenesis (Wat-

tenberg, 1990; Kelloff et al., 1994; Morse and

Stoner, 1993; Stoner and Mukhtar, 1995). The

intervention of cancer at the promotion stage,

however, seems to be the most appropriate and

practical. The major reason for that relates to the

fact that tumor promotion is reversible event at

least in early stages and requires repeated and

prolonged exposure of a promoting agent (DiGio-

vanni, 1992).

The administration of the carcinogen BaP

caused 100% incidence of forestomach tumors,

while the oil treatment did not induce any tumors

in the recipient animals. The tumor multiplicity in

the BaP alone group was also high (6.24). A

similar effect of BaP in tumor induction has been

reported earlier (Wattenberg et al., 1980; Nagab-

hushan and Bhide, 1987; Azuine and Bhide, 1992;

Deshpande et al., 1997; Agha et al., 2001). The

administration of ASE as sole source of drinking

water before, during and after the carcinogen

Table 2

Effect of different administration schedules on the chemopreventive activity of 4 mg/ml ASE in the BaP-induced forestomach

carcinoma in mice

Drug dose (mg/ml) Carcinogen (8 mg) Tumor incidence Mean number Tumor multiplicity Mean body weight (g)

Initial weight Final weight

ASE 0 pre�/post DDW 0 (0/5) �/ �/ 22.119/0.85 32.129/0.92

ASE 0 pre�/post Oil only 0 (0/5) �/ �/ 21.949/0.92 32.259/0.87

ASE 0 pre�/post Oil�/BaP 100 (20/20) 7.0 (100) 6.2 (100) 22.459/0.56 28.869/1.36

ASE 4 pre only Oil only 0 (0/5) �/ �/ 22.679/0.84 32.219/0.98

ASE 4 pre only Oil�/BaP 100 (15/15) 6.5 (92.85)d 6.1 (98.38)d 22.639/0.92 30.419/1.08b

ASE 4 post only Oil only 0 (0/5) �/ �/ 22.079/0.84 31.989/0.98

ASE 4 post only Oil�/BaP 100 (15/15) 6.5 (92.85)d 5.7 (91.93)d 22.239/0.92 30.749/1.08c

ASE 4 pre�/post Oil only 0 (5/5) �/ �/ 22.679/0.84 34.219/0.98

ASE 4 pre�/post Oil�/BaP 93.33 (14/15)d 3.5 (50)b 3.8 (61.29)b 22.189/0.83 31.759/1.11c

a, P B/0.01; b, P B/0.02; c, P B/0.001; d, non significant (comparisons done with BaP treated group). Number in parentheses in

column 3 indicates number of animals with tumor/total number of animals used, while in column 4 and 5 denotes percent decrease in

the occurrence of tumors when compared with the concurrent BaP alone group.

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193188

treatment suppressed the BaP-induced foresto-

mach tumorigenesis. There was a dose dependent

decrease in the multiplicity of the tumors and the

highest effect was observed for 4 mg/ml ASE,

where the occurrence was lesser than the other two

doses. As far as the authors are aware this is the

first report on the alterations in the chemically-

induced carcinogenesis by ASE.

The second aspect of the study was to ascertain

whether delay in the administration of 4 mg/ml of

ASE, i.e. starting from 48 h after the last dose of

BaP for 8 weeks will also be effective in inhibiting

the formation of multiple gastric tumors induced

by the carcinogen. Our findings indicate that the

ASE can also act at this stage and has been able to

counteract the tumorigenesis as evident by the

reduced multiplicity (5.78) of the tumor when

compared with the BaP alone group (6.24). Inspite

of this reduced multiplicity, the post-treatment of

ASE was not as effective as the pre�/post-treat-

ment regimen. A similar observation has been

reported for the turmeric and its extracts (Desh-

pande et al., 1997).

We were also interested to know whether the

administration of 4 mg/ml of ASE when adminis-

tered continuously for 2 weeks before the admin-

istration of BaP will also inhibit the BaP-induced

tumorigenesis? Our results show that the drug was

ineffective with this schedule and the tumor

incidence remained unaltered. However, the tumor

multiplicity was marginally reduced to 6.13 when

compared with BaP alone group (6.24). This

regime was ineffective when compared with the

pre�/post and post-treatment regimen.These results are supported by the studies on the

micronucleus-induction, which is an index of

DNA damage. The frequency of MN increased

4-folds in the splenocytes of animals treated with

BaP for forestomach carcinogenesis. BaP has been

observed to be an inducer of MN in the spleen

(Benning et al., 1994; Winker et al., 1995; Dertin-

ger et al., 2001). While the treatment of mice with

different doses of ASE before and after signifi-

cantly reduced the frequency of MN, indicating

reduction in the BaP-induced DNA damage. This

may be one of the reasons for the decline in tumor

incidence in pre- and post-ASE treated group.Ta

ble

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G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193 189

The formation of DNA adducts inflicts loss of

nuclear functioning that leads to mutations and

chromosome aberrations. The mutational events

and chromosomal instability are key steps in

carcinogenesis and many types of cancer have

been associated with the chromosomal aberrations

(Heim and Mitelman, 1987; Heim et al., 1989).

Damage to the chromosomes is expressed as

breaks and fragments which, appear as MN in

the proliferating cells (Heddle and Harris, 1975).

The chromatid breaks and exchanges that lead to

chromosome anomalies, must occur during carci-

nogenesis to attain the neoplastic property. The

tumor cell population is composed of one or few

clones of cells with particular chromosome anoma-

lies and this phenomenon is detectable by an

increased frequency of MN in the proliferating

cells (Countryman and Heddle, 1976; Heim and

Mitelman, 1989). The cell proliferation also allows

endogenous adducts to be converted into muta-

tions and makes more sensitive single-stranded

DNA target for carcinogenesis. Therefore, the

evaluation of MN gives an indication on the

quantitative aspect on the insult to the cellular

DNA. In chronic administration studies, it is

always desirable to study the MN formation in

the spleen, because the irreparable damage to the

cellular DNA gets accumulated, which becomes

manifest when the splenocytes are stimulated to

divide. BaP has been reported to be immunotoxic

and has profound inhibitory effect on the spleen

(Ginsberg et al., 1989; Zhao, 1990). Earlier reports

indicate that the BaP treatment resulted in an

increase in the BaP�/DNA adduct formation and

frequency of mutation in the spleen cells (Benning

et al., 1994; Winker et al., 1995; Dertinger et al.,

2001).The exact mechanism of the chemopreventive

effect of ASE is not known. However, the opera-

tions of these mechanisms cannot be ruled out.

Alterations in the native DNA play an impor-

tant and crucial role in carcinogenesis. The neo-

plastic cells contain high frequencies of

chromosomal aberrations. The treatment of mice

with ASE may have reduced the DNA damage as

evident by a significant reduction in the frequency

of MN. Therefore, protection against the DNA

damage by ASE during tumorigenesis may be oneof the reasons for lesser frequency of tumors.

In this study we have observed that the chemo-

preventive effect was the best when ASE is

administered during and after (pre�/post) the BaP

treatment and this effect may be because of the

involvement of the Phase I and Phase II metabo-

lizing enzymes. A. scholaris has been reported to

inhibit the liver injuries induced by carbon tetra-chloride, beta-D-galactosamine, acetaminophen

and ethanol in mice and rats (Lin et al., 1996).

These agents like BaP are all activated to reactive

forms by the cytochrome P450 (Mehendale, 1995;

Ray and Mehendale, 1990; Thapliyal and Maru,

2001) and the efficacy of ASE in inhibiting the

damage induced by these agents strongly suggests

that the ASE may have down regulated orinhibited the action of cytochrome P450 and

thereby reducing the occurrence of BaP-induced

tumors in the present study.

In our earlier study we have observed that the

ASE was highly effective as a nitric oxide (NO)

scavenger (data not shown). This activity of ASE

may also have been responsible for the chemopre-

ventive activity. A medline study also shows thatthe BaP induces synthesis of NO and its progeni-

tors in vitro and in vivo (Garcon et al., 2001;

Takabe et al., 2001) and the compounds that are

inhibitors like Ginkgo biloba , curcumin, triterpe-

noids, resveratrol, nimesulide, L-nitroarginine

methyl ester and sulforaphane have been observed

to be chemopreventive in different study systems

(Li and Lin-Shia, 2001; Suh et al., 1998; Rao et al.,1999; Lin and Tsai, 1999; Watanabe et al., 2000;

Sharma et al., 2002; Kawamori et al., 2000; Heiss

et al., 2001; Agha et al., 2001).

The ASE contains triterpenoids (Lupeol linole-

ate, lupeol palmitate and alpha-amyrin linoleate) a

class of compounds reported to inhibit the serine

proteases and PKA (Rajic et al., 2000). The

protease inhibitors have been reported to inhibitcarcinogenesis in different study systems and their

mechanism of action is postulated to be due to

suppression of the neoplastic cells and tumor

promotion (Tanaka, 1994). The presence of triter-

penoids in the ASE may also have contributed to

its chemopreventive property. This contention is

supported by earlier observations, where triterpe-

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193190

noids tubeimoside, abieslactone, 3beta-cis -p -cou-maroyloxy-2alpha, tubeimosides and 23-dihydrox-

yolean-12-en-28-oic acid have been reported to be

chemopreventive in different study systems (Yu et

al., 1992; Suh et al., 1999; Yu et al., 2001; Gu et al.,

2001; Iwai et al., 2001).

The immune system plays an important role in

the maintenance of health. The basic concept of

immunoserveillance is that the development ofcancer cells in the organism stimulates a potent

immune response that locates the cancer cells and

destroys them (Shklar, 1998). A. scholaris has

recently been reported to possess immunostimula-

tory effect and also to enhance the phagocytic

activity in normal as well as in the immunosup-

pressed mice (Iwo et al., 2000). Therefore, the

immunomodulation may also have played somerole in the observed chemopreventive activity of

ASE.

Alstonia has been reported to possess alkaloid

echitamine, which has been reported to be selec-

tively cytotoxic to the tumor cells (fibrosarcoma

and in sarcoma-180), while it had no effect on the

normal cells (Kamarajan et al., 1995; Saraswathi et

al., 1997). Echtamine is also present in the extract(since it is isolated from the ethanolic extract), and

may have inhibited the tumor growth by selec-

tively killing the aberrant cells. Further, the drug

comes in direct contact with the target site of

tumorigenesis so a similar action of ASE in the

present study may not be ruled out. The studies are

underway to identify the active component(s)

responsible for the chemoprotective propertieswith emphasis on the study of their mechanism(s)

of action.

Acknowledgements

We would like to thank Dr G.B. Maru, Head

Tobacco Carcinogenesis Division, Cancer Re-

search Institute, ACTREC, Tata memorial Centre,Kharghar, Mumbai, India for his constant support

and guidance throughout the study. The authors

wish to thank Professor Gopalkrishna Bhat,

Poorna Prajna College Udupi, Karnataka, for

identifying the plant material. The financial assis-

tance in the form of a Senior Research Fellowship

to M.S. Baliga by the Indian Council for MedicalResearch, Government of India, New Delhi, India

to carry out this study is thankfully acknowledged.

References

Agha, A.M., El-Fattah, A.A., Al-Zuhair, H.H., Al-Rikabi,

A.C., 2001. Chemopreventive effect of Ginkgo biloba extract

against benzo(a)pyrene-induced forestomach carcinogenesis

in mice: amelioration of doxorubicin cardiotoxicity. J. Exp.

Clin. Cancer Res. 20, 39�/50.

Azuine, M.A., Bhide, S.V., 1992. Chemopreventive effect of

turmeric against stomach and skin tumors induced by

chemical carcinogens in Swiss mice. Nutr. Cancer 17, 77�/

83.

Benning, V., Brault, D., Duvinage, C., Thybaud, V., Melcion,

C., 1994. Validation of the in vivo CD1 mouse splenocyte

micronucleus test. Mutagenesis 9, 199�/204.

CHEMXCIL, 1992. Selected Medicinal Plants of India. Basic

chemicals, Pharmaceutical and Cosmetic Export Promotion

Council, Bombay 400 039, India.

Countryman, P.I., Heddle, J.A., 1976. The production of

micronuclei from chromosome aberrations in irradiated

cultures of human lymphocytes. Mutat. Res. 41, 321�/332.

Dertinger, S.D., Nazarenko, D.A., Silverstone, A.E., Gasie-

wicz, T.A., 2001. Aryl hydrocarbon receptor signaling plays

a significant role in mediating benzo[a]pyrene- and cigarette

smoke condensate-induced cytogenetic damage in vivo.

Carcinogenesis 22, 171�/177.

Deshpande, S.S., Ingle, A.D., Maru, G.B., 1997. Inhibitory

effects of curcumin-free aqueous turmeric extract on

benzo[a]pyrene-induced forestomach papillomas in mice.

Cancer Lett. 16, 79�/85.

DiGiovanni, J., 1992. Multistage carcinogenesis in mouse skin.

Pharmacol. Ther. 54, 63�/128.

Fenech, M., Morley, A.A., 1985. Measurement of micronuclei

in lymphocytes. Mutat. Res. 147, 29�/36.

Gandhi, M., Vinayak, V.K., 1990. Preliminary evaluation of

extracts of Alstonia scholaris bark for in vivo antimalarial

activity in mice. J. Ethnopharmacol. 29, 51�/57.

Garcon, G., Gosset, P., Garry, S., Marez, T., Hannothiaux,

M.H., Shirali, P., 2001. Pulmonary induction of proinflam-

matory mediators following the rat exposure to BaP-coated

onto Fe2O3 particles. Toxicol. Lett. 30, 107�/117.

Ginsberg, G.L., Atherholt, T.B., Butler, G.H., 1989. Ben-

zo[a]pyrene-induced immunotoxicity: comparison to DNA

adduct formation in vivo, in cultured splenocytes and in

microsomal systems. J. Toxicol. Environ. Health 28, 205�/

220.

Gu, J.Q., Park, E.J., Luyengi, L., Hawthorne, M.E., Mehta,

R.G., Farnsworth, N.R., Pezzuto, J.M., Kinghorn, A.D.M.,

2001. Constituents of Eugenia sandwicensis with potential

cancer chemopreventive activity. Phytochemistry 58, 121�/

127.

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193 191

Halliwell, B., Gutteridge, J.M., 1989. Free Radicals in Biology

and Medicine, second ed. New York Oxford University,

Claredon Press, USA, pp. 466�/495.

Heddle, J.A., Harris, J.W., 1975. Letter: rapid screening of

radioprotective drugs in vivo. Radiat. Res. 61, 350�/353.

Heim, S., Mitelman, F., 1987. Ninteen of 26 cellular oncogenes

precisely localized in the human genome map to one of the

83 bands involved in primary cancer specific rearrange-

ments. Hum. Genet. 75, 70�/72.

Heim, S., Mitelman, F., 1989. Primary chromosome abnorm-

alities in human neoplasia. Adv. Cancer Res. 52, 1�/43.

Heim, S., Johansson, B., Mertens, F., 1989. Constitutional

chromosome instability and cancer risk. Mutat. Res. 221,

39�/51.

Heiss, E., Herhaus, C., Klimo, K., Bartsch, H., Gerhauser, C.,

2001. Nuclear factor kappa B is a molecular target for

sulforaphane-mediated anti-inflammatory mechanisms. J.

Biol. Chem. 24, 32008�/32015.

Iwai, S., Wei, M., Morimura, K., Wanibuchi, H., Tanaka, R.,

Matsunaga, S., Yoshitake, A., Seki, S., Fukushima, S.,

2001. Possible prevention by abieslactone of development of

diethylnitrosamine-initiated GST-P positive foci in the rat

liver. Teratog. Carcinog. Mutag. 21, 223�/229.

Iwo, M.I., Soemardji, A.A., Retnoningrum, D.S., Sukrasno,

U.M., 2000. Immunostimulating effect of pule (Alstonia

scholaris L. R.Br., Apocynaceae) bark extracts. Clin.

Hemorheol. Microcirc. 23, 177�/183.

Jagetia, G.C., Menon, K.S., Jain, V., 2001. Genotoxic effect of

hydroquinone on the cultured mouse spleenocytes. Toxicol.

Lett. 8, 15�/20.

Kamarajan, P., Ramamurthy, N., Govindasamy, S., 1995. In

vitro evaluation of the anti-cancer effects of echitamine

chloride on fibrosarcoma cells. J. Clin. Biochem. Nutr. 18,

65�/71.

Kawamori, T., Takahashi, M., Watanabe, K., Ohta, T.,

Nakatsugi, S., Sugimura, T., Wakabayashi, K., 2000.

Suppression of azoxymethane-induced colonic aberrant

crypt foci by a nitric oxide synthase inhibitor. Cancer

Lett. 1 (148), 33�/37.

Keawpradub, N., Houghton, P.J., Eno-Amooquaye, E., Burke,

P.J., 1997. Activity of extracts and alkaloids of thai Alstonia

species against human lung cancer cell lines. Planta Med. 63,

97�/101.

Keawpradub, N., Eno-Amooquaye, E., Burke, P.J., Houghton,

P.J., 1999. Cytotoxic activity of indole alkaloids from

Alstonia macrophylla . Planta Med. 65, 311�/315.

Kelloff, G.J., Boone, C.W., Steele, V.E., Crowell, J.A., Lubet,

R., Sigman, C.C., 1994. Progress in cancer chemopreven-

tion: perspectives on agent selection and short-term clinical

intervention trials. Cancer Res. 1 (54), 2015s�/2024s.

Krisch-Volders, M., Souni, T., Aardema, M., Albertini, S.,

Eastmond, D., Fenech, M., Ishidate, M., Jr, Lorge, E.,

Norppa, H., Surralles, J., Von Dur Hude, W., Wakata, A.,

2000. Report from the in vitro micronucleus assay-working

group. Environ. Mol. Mutagen. 35, 167�/

172.

Li, J.K., Lin-Shia, S.Y., 2001. Mechanisms of cancer chemo-

prevention by curcumin. Proc. Natl. Sci. Counc. Repub.

Chin. B 25, 59�/66.

Lin, J.K., Tsai, S.H., 1999. Chemoprevention of cancer and

cardiovascular disease by resveratrol. Proc. Natl. Sci.

Counc. Repub. Chin. B 23, 9�/16.

Lin, S.C., Lin, C.C., Lin, Y.H., Supriyatna, S., Pan, S.L., 1996.

The protective effect of Alstonia scholaris R. Br. on

hepatotoxin-induced acute liver damage. Am. J. Chin.

Med. 24, 153�/164.

Mehendale, H.M., 1995. Toxicodynamics of low level toxicant

interactions of biological significance: inhibition of tissue

repair. Toxicology 28, 251�/266.

Morse, M.A., Stoner, G.D., 1993. Cancer chemoprevention:

principles and prospects. Carcinogenesis 14, 1737�/1746.

Nadkarni, A.K., 1976. Indian Materia Medica, third ed.

Popular Press, Mumbai, India.

Nagabhushan, M., Bhide, S.V., 1987. Antimutagenicity and

anticarcinogenicity of turmeric (Curcuma longa ). J. Nutr.

Growth Cancer 4, 83�/89.

Rajic, A., Kweifio-Okai, G., Macrides, T., Sandeman, R.M.,

Chandler, D.S., Polya, G.M., 2000. Inhibition of serine

proteases by anti-inflammatory triterpenoids. Planta Med.

66, 206�/210.

Rao, C.V., Kawamori, T., Hamid, R., Reddy, B.S., 1999.

Chemoprevention of colonic aberrant crypt foci by an

inducible nitric oxide synthase-selective inhibitor. Carcino-

genesis 20, 641�/644.

Rastogi, R.M., Mehrotra, B.N., 1990. Compendium of Indian

Medicinal Plants, vol. 1. Central drug research Institute,

Lucknow, India, pp. 388�/389.

Ray, S.D., Mehendale, H.M., 1990. Potentiation of CCl4 and

CHCl3 hepatotoxicity and lethality by various alcohols.

Fundam. Appl. Toxicol. 15, 429�/440.

Saraswathi, V., Ramamurthy, N., Subramaniam, S.,

Mathuram, V., Govindasamy, S., 1997. Enhancement of

the cytotoxic effects of echitamine chloride by vitamin A: an

in vitro study on Ehrlich ascites carcinoma cell culture.

Indian J. Pharmacol. 29, 244�/249.

Satyavati, G.V., Gupta, A.K., Tandon, N., 1987. Medicinal

Plants of India, vol. 2. Indian Council of Medical Research,

New Delhi, India, pp. 230�/239.

Sharma, S., Wilkinson, B.P., Gao, P., Steele, V.E., 2002.

Differential activity of NO synthase inhibitors as chemo-

preventive agents in a primary rat tracheal epithelial cell

transformation system. Neoplasia 4, 332�/336.

Shklar, G., 1998. Mechanisms of cancer inhibition by anti-

oxidant nutrients. Oral Oncol. 34, 24�/29.

Stoner, G.D., Mukhtar, H., 1995. Polyphenols as cancer

chemopreventive agents. J. Cell. Biochem. Suppl. 22, 169�/

180.

Suh, N., Honda, T., Finlay, H.J., Barchowsky, A., Williams,

C., Benoit, N.E., Xie, Q.W., Nathan, C., Gribble, G.W.,

Sporn, M.B., 1998. Novel triterpenoids suppress inducible

nitric oxide synthase (iNOS) and inducible cyclooxygenase

(COX-2) in mouse macrophages. Cancer Res. 15 (58), 717�/

723.

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193192

Suh, N., Wang, Y., Honda, T., Gribble, G.W., Dmitrovsky, E.,

Hickey, W.F., Maue, R.A., Place, A.E., Porter, D.M.,

Spinella, M.J., Williams, C.R., Wu, G., Dannenberg, A.J.,

Flanders, K.C., Letterio, J.J., Mangelsdorf, D.J., Nathan,

C.F., Nguyen, L., Porter, W.W., Ren, R.F., Roberts, A.B.,

Roche, N.S., Subbaramaiah, K., Sporn, M.B., 1999. A

novel synthetic oleanane triterpenoid, 2-cyano-3, 12-diox-

oolean-1,9-dien-28-oic acid, with potent differentiating,

antiproliferative, and anti-inflammatory activity. Cancer

Res. 15, 336�/341.

Takabe, W., Niki, E., Uchida, K., Yamada, S., Satoh, K.,

Noguchi, N., 2001. Oxidative stress promotes the develop-

ment of transformation: involvement of a potent mutagenic

lipid peroxidation product, acrolein. Carcinogenesis 22,

935�/941.

Tanaka, T., 1994. Cancer chemoprevention by natural pro-

ducts. Oncol. Rep. 1, 1139�/1155.

Thapliyal, R., Maru, G.B., 2001. Inhibition of cytochrome

P450 isozymes by curcumins in vitro and in vivo. Food

Chem. Toxicol. 39, 541�/547.

Warrier, P.K., Nambiar, V.P.K., Ramankutty, C., 1996. Indian

Medicinal Plants, vol. 5. Orient Longman Ltd, Hyderabad,

India, pp. 225�/228.

Watanabe, K., Kawamori, T., Nakatsugi, S., Wakabayashi, K.,

2000. COX-2 and iNOS, good targets for chemoprevention

of colon cancer. Biofactors 12, 129�/133.

Wattenberg, L.W., 1990. Inhibition of carcinogenesis by minor

anutrient constituents of the diet. Proc. Nutr. Soc. 49, 173�/

183.

Wattenberg, L.W., Coccia, J.B., Lam, L.K., 1980. Inhibitory

effects of phenolic compounds on benzo(a)pyrene-induced

neoplasia. Cancer Res. 40, 2820�/2823.

Winker, N., Weniger, P., Klein, W., Ott, E., Kocsis, F.,

Schoket, B., Korpert, K., 1995. Detection of polycyclic

aromatic hydrocarbon exposure damage using different

methods in laboratory animals. J. Appl. Toxicol. 15, 59�/

62.

Yu, L.J., Ma, R.D., Wang, Y.Q., Nishino, H., Takayasu, J.,

He, W.Z., Chang, M., Zhen, J., Liu, W.S., Fan, S.X., 1992.

Potent antitumorigenic effect of tubeimoside 1 isolated from

the bulb of Bolbostemma paniculatum (Maxim) Franquet.

Int. J. Cancer 20, 635�/638.

Yu, T.X., Ma, R.D., Yu, L.J., 2001. Structure-activity relation-

ship of tubeimosides in anti-inflammatory, antitumor, and

antitumor-promoting effects. Acta Pharmacol. Sin. 22,

463�/468.

Zhao, X.L., 1990. Effects of benzo(a) pyrene on the humoral

immunity of mice exposed by single intraperitoneal injec-

tion. Zhonghua. Yu. Fang. Yi. Xue. Za. Zhi. 24, 220�/

222.

G.C. Jagetia et al. / Toxicology Letters 144 (2003) 183�/193 193