Chapter IV

Pharmacological activities of Picrolv

Section 1

Antioxidant and Antiturnour activity of Picroliv

Antioxidant and antiturnour activity of Picroliv

1. Introduction

The involvement of oxidative stress in cancer induction and associated molecular

mechanisms is becoming increasingly clear. There is abundant evidence that activated

oxygen species are potentially involved in initiation, promotion and'progression stage of

carcinogenesis. The scavengers of reactive carcinogens, notably the antioxidant agents,

have been the subject of an increasing number of investigators into protective mechanisms

for various cancers in both experimental animal studies and human epidemiological studies

.There is direct evidence that scavenging of activated oxygen species is a mechanism for

inhibiting carcinogenesis. Efforts, therefore, are being made to identify naturally occumng

antioxidants which would prevent, slow andlor reverse the cancer induction and its

subsequent development

Picrorhiza kurroa (Regional name Kutki) forms an ingredient of many Indian

herbal preparations used for the treatment of liver ailments. Picroliv is the standard

preparation containing mainly a mixture of two iridoid glycosides, picroside-1 and

kutkoside (1: 1.5 wlw, Fig 1.5) purified from the e.thanolic extract of the roots and rhizomes

of the Picrorhiza kurroa (P. kurroa). Picroliv has been reported to be a potent

hepatoprotective agent against various hepatotoxins including hepatitis B virus (Dwivedi et

al., 1990; Dwivedi et al., 1993; Dhawan, 1995). Picroliv has been shown to scavenge

superoxide anions (Chander et al., 1992 a) and induce glutathione S-transferase (Rastogi et

al., 1995). In addition, it could reduce the increased levels of lipid peroxidation products

induced by Plasmodium berghei in liver and brain of African desert rat (Mastomys

natalensis) resulting in recovery of reduced glutathione levels and the activities of

glutathione-related enzymes (Chander et al., 199:! b; Chander et al., 1994).

The present section focuses attention on the antioxidant activity of Picroliv. Studies

were also conducted to assess the effect of Picroliv treatment on ascites and solid tumour

development induced by transplantable tumou~rs in mice. In addition, it explores the

effic:acy of Picroliv on inhibiting aniline hydroxylase, DNA topoisomerase and cdc25

tyrosine phosphatase.

2. Materials and methods

2.1. Determination of in vitro antioxidant activity of Picroliv

Superoxide scavenging activity of Picroliv was determined by the light induced

superoxide generation by riboflavin and subsequent reduction of nitrobluetetrasolium as

described by Mc Cord and Fridovich (1969).The procedure is given in chapter 11.

Different concentrations of Picroliv (10-60 pglml) was dissolved in the reaction mixture

and concentration needed for 50 % inhibition was calculated.

Hydroxyl radical scavenging activity of Picroliv was measured by studying the

competition between deoxyribose and the test co~npounds for hydroxyl radicals generated

from the Fe " 1 ascorbate / EDTA / H202 system. 'The hydroxyl radicals attack deoxyribose

which eventually results in the formation of thiobarituric acid reacting substances which

was estimated by the method descried by Ohkawa et a1 (1979). The procedure is given in

chapter 11. The inhibition produced by different concentrations of Picroliv (2-12 pg/ml) as

well as the concentration required for 50 % inhibition was calculated.

Lipid peroxidation was induced in rat liver homogenate by the method described by

Bishayee and Balasuramonanian (1971) in the presence of different concentrations of

Picroliv (200-1200 pglml) and measured by the method described Ohkawa et a1 (1979).

The procedure has given in chapter 11. The percl-ntage inhibition was calculated and the

concentration requ~red for 50 % inhibilion was calculated.

2.2. Determination of the effect of Picroliv on aniline hydroxylase

Aniline hydroxylase assay was performed by the method described by Mazel

(Maze1,1971). The enzyme was induced in rats by the oral administration of phenobarbital

(80 mg/kg) for 5 continous days. A 10 % liver homogenate prepared in 10 rnM ice cold

tris-HCI buffer (pH 7.4) containing 0.25M sucrose was used for assay. P-aminophenol

formed during the enzyme action reacts with phenol in alkaline medium to form a blue

coloured product, which was measured at 630 nm. The percentage inhibition of aniline

hydroxylase was calculated by comparing the absorbency of control and that of drug

treated samples.

2.3. Determination of the effect of Picroliv on Topoisomerase I and 11.

The DNA topoisomerases are a group of enzymes that alter DNA topology by

causing and resealing DNA strand breaks. Saccharomyces cervrsiae mutant cell cultures

JN 394, JN 394 , and JN 394 ,.2.5 were used for topoisomerase inhibitory assays. The

organisms were cultured in petridishes containing YPDA medium (20 ml). The cells from

a fully grown plate of each organisms were suspended in saline solution (10 ml) and then

diluted to obtain 5 X 10 CFUIml. 50 p1 of this suspension was then used to inoculate

petridishes containing YPDA media and allowed to air dry in the laminar flow hood for 20

minutes. Picroliv was dissolved in DMSO and added to the inoculated plates (20 p1) to

give a final concentration of 250 pgl ml. These plates were inoculated at 2 7 ' ~ for 72-96 h.

At the end of incubation period the zones of inhibition were recorded for each test

organism. Controls were prepared by adding DMSO (20 p1) to inoculated plate (Chang et

al., 1995; Roth et al., 1998)

2.4. Determination of the effect of Picroliv on cdc25 tyrosine phosphatase.

20 pl of GST-cdc25A protein was mixed with 20 p1 100 rnM dithiothreitol in Tris

buffer. Different concentrations of Picroliv was dissolved in Tris buffer (140 pl), in 96-

well microtitration plates. The plates were preincubated at 37' C for 15 minutes in a

Denley W e l l w m I microplate incubator. The assay was initiated by addition of 20 p1 of

500 mM p-nitrophenylphosphate phosphatase (p-NPP). After 60 min incubation at 37OC

absorbance at 405 nm was measured in a BioKad microplate reader. Blank values (no

GST-cdc25A protein but 10 minutes incubation with substrate) were automatically

substracted (Baratte et al., 1992).

2.5. Determination of the effect of Picroliv on ascites tumour development induced by

transplanted tumours

Tumour cells (DLA I EAC) aspirated from the peritoneal cavity of mice were

washed thrice with saline and 1 x 10 tumour cells were given intraperitoneally to four

group of animals (10-weeks-old, 25 g, male Swiss albino mice, 7 micelgroup. Animals in

the group I was kept as control mice with out drug treatment. 24 h after tumour inoculation

animals in groups 11-IV received Picroliv at a concentration of 15, 75, 375 mg/kg body

weight (p.o), respect~vely, and continued daily for 10 days.

Animals were observed for the development of ascites tumour and deaths due to

tumour burden were recorded. The increase in life span (percent ILS) of treated group was

calculated using the formula, percent I L S = (T - C) I C x 100, where 'T and 'C are mean

survival of treated and control mice, respectively (Soudhamini and Kuttan, 1988).

2.6. Determination of the effect of Picroliv on solid tumour development induced by

transplanted tumours

One million DLA I EAC cells were injected in to the right hind limb of male Swiss

albino mice (10-weeks-old, 25 g, 7 micelgroup). Animals in the group I was kept with out

drug, treatment. 24 h after tumour inoculation anirnals in groups 11-IV received Picroliv at a

concentration of 15, 75, 375 mglkg body weight (p.o), respectively, and continued for 10

days. Tumour diameter was measured on every fifth day using vernier calipers and volume

was calculated uslng the formula, volume = 4/3 r rl x r2, where rl and r2 represents the

radir of the tumour at two different planes. The survival of the animals was recorded for 60

days.

3. Results

3.1. Antioxidant activity of Picroliv

P~croliv was found to scavenge superoxrdes and hydroxyl radicals and inhibited

lipid peroxldation in vltro (Fig IV.l.l) The concentration of Picroliv required for 50 %

scavenging of superoxide generation was found to be 39 p g / d (Fig IV.I.1). The

concentration of known antioxidants such as ellagic acid and curcumin needed for the

same effect were 7.5 and 9.2 pgtml, respectively (Jose and Kuttan, 1995). Picroliv also

effectively scavenged degradation of deoxyribocie mediated by hydroxyl radicals formed

during Fenton's reaction. Concentration required for 50 % scavenging of hydroxyl radical

was found to be 8.8 pg/ml (Fig IV. I . I). The concentration required for 50 % scavenging of

the hydroxyl radical formation was reported to be 27.6 C l g / d (Elizabeth and Rao, 1990).

Similarly, Picroliv inhibited generation of lipid peroxides by ~ e ~ + / ascorbate in rat liver

homogenate. The concentration required for 50 % inhibition was found to be 880 pglml

(Fig IV.I.l). The concentration required for 50 % inhibition of lipid peroxidation by

curcumin and ellagic acid were reponed to be 7.4 and 45.3 pglml, respectively (Jose and

Kuttan, 1995).

3.2. Effect of Picroliv on aniline hydroxylase, topoisomerase I and I1 and cdc25

tyrosine phosphatase

Picroliv was found to be ineffective in inhibiting aniline hydroxylase. Picroliv at a

concentration of 500 ~ g / m l produced only 11 % inhibition of aniline hydroxylase.

Similarly, Picroliv was found to be ineffective in inhibiting cdc25 tyrosine phosphatase.

Fig. IV.I.1 Antioxidant activity of Picroliv

Concentration -- of Picroliv @g/mL)

A- Lipid Peroxidation, + - Super oxide radical

- Hydroxyl radical

Concentration required for 50 % inhibition was found to be above 1000 pglml. Picroliv

was found to inhibit the activity of topoisomerase I1 of Saccharomyces cervisiae at a

concentration of 250 pglml. However, this concentration was found to be ineffective in

inhibiting topoisomerase I.

3.3. Effect of Picroliv on ascites tumour development

Administration of Picroliv was found to increase the life span of ascites tumour

bearing mice. All the untreated animals (Plate 4 c) in the DLA tumour group died of

tumour burden 22.5 + 2.4 days after tumour inoculation where as the Picroliv treated

animals (Plate 4 d) survived 23.4 + 3.3, 31.1 + 5.1 and 37.5 + 5.4 days in 15, 75 and 375

mgkg, respectively (Table IV.1. I). The entire untreated EAC tumour inoculated mice died

of tumour burden with in 16.5 k 2.0 days. Picroliv treatment increased the survival to 6.8,

22.3 and 51.7 %, respectively at the same concentrations of Picroliv (Table N.l.l).

3.4. Effect of Picroliv on solid tumour development

Administration of Picroliv reduced the tumour volume of both DLA and EAC cell

lines in a dose dependent way. The tumour volume of untreated mice (Plate 4 a) on 30 th

day after tumour (DLA) inoculation was found to be 8.4 cc. The tumour volume (Plate 4 d)

was reduced to 6.4, 5.0, and 3.0 cc by Picroliv administration at concentrations of 15, 75

and 375 mgkg body weight, respectively (Fig 1V.1.2). Similarly, the tumour volume of

EAC: inoculated animals on 3 0 ~ day after tumour inoculation was found to be 8.6 cc which

was reduced to 7.0, 5.7 and 2.6 cc and the percent reduction in tumour volume was found

to be 19.0,33.7 and 69.5 % in the group of animals treated with Picroliv (Fig IV.1.2).

4. Discussion

The involvement of oxygen-derived free radicals in the pathophysiology of many

human diseases has led to considerable research :into pharmacological antioxidants for the

treatment and prevention of disease including cancer. In this regard, the efficacy of

Picroliv in scavenging superoxide and hydroxyl radicals generated in in vitro system were

studied. The present study showed that Picroliv icould scavenge superoxide and hydroxyl

radic:als. The concentration required for 50 % scavenging of hydroxyl radical by Curcumin

was reported to be 27.6 pg/ml (Elizabath and Rao., 1990), but in the case of Picroliv the

concentration required for 50 % scavenging of hydroxyl radical was found to be 8.8

pglml, indicating the strong antioxidant potential of Picroliv. This shows that Picroliv is

Plate 4

Mouse showing transplanted solid tumoul-s, sarcomas and

papillomas

(a) Solid turnour bearing mize inoculated with DEA tumour showing

increased tunlour volume.

(b) Solid tumour bearing mouse treated with Picroliv 375 mg'kg showing

reduction in turnour volume.

(c) Ascites turnour bearing mice inoculated with DLA cell lines.

(d) Ascites turnour mice treated with Picroliv 375 mgkg.

(e) Mice which developed sarcomas induced by 20-MC.

(f) Sarcoma bearing mice treated with Picroliv 150 mgkg.

(g) Mouse showing papillomas induced by DMBA and croton cii!.

(h) Mouse treated with Picroliv 5 mgldose shows reductixl iil the number 3f

papil lo~~as.

Table IV. 1 . 1 . Effect of P'icroliv administration on the survival of ascites tumour

harboring mice inoculated with DLAiEAC cell lines

Group

I

I1

111

IV

a = p < 0.001, c = p < 0.01 as compared to group I. Values are mean SD, n = 7

Animal status

Wlthout drug

P ~ c r o l ~ v 15 mg/ky

Picroliv 75 mg/kg

Plcrollv 375 mgkg

DLA tumour

Average life span (days)

22.5'1 * 2.49

23.4;! * 3.37

31.14*5.15

37.57 i 5.42 a

EAC tumour

% increase in life span

0

3.76

37.97

66.45

Average life span (days)

16.57 * 2.00

17.71 i 2.71

20.18 * 3.76

25.14 * 5.84

% increase in life span

0

6.87

22.38

51.71

Fig. IV.1.2. The effect of Picroliv treatment on solid tumour volumes induced by DWEAC cell line in mice.

EAC tumour

Days post tumour inoculation

- Tumour cells alone; A - Picroliv 15 mglkg; . - Picroliv 75mglkg; 0 - Picroliv 375 mglkg

superior to Curcumin in scavenging hydrox:yl radicals. However, Curcumin could

scavenge superoxide radicals relatively small concentration as compared to Picroliv. The

concentrations required for 50 % scavenging of superoxide radicals by Curcumin was 9.2

pg/rnl (Jose and Kuttan, 1995) and that of Picrol:iv was found to be 39 pglml.

Lipid peroxidation inflicts cell damage whenever conditions of increased oxidative

stress occur in the cell (Dargel, 1992). Reactive products after lipid peroxidation, may

function as co-carcinogenic agents by being highly cytotoxic and inhibits enzyme

functions such as DNA repair (Krokan et a]., 1!385). Recently free radical induced lipid

peroxidation has gained much importance because of its involvement in several

pathologies including cancer (Kumar et al., 1991)1. Protection of cell membranes from lipid

peroxidation becomes a necessity to prevent, cure or delay the tissue damage. MDA levels

are widely used as marker of free radical induced lipid peroxidation injury. Higher levels

of MDA has been reported in coiorectal and breast cancer patients (Otamiri and Sjodahl,

1989; Kumar et al., 1991) Our study shows that Picroliv could inhibit the lipid

peroxidation in rat liver homogenate induced by F;e 2 + ascorbate system. Similar protection

against lipid peroxidation by Picroliv has been reported by other investigators (Rastogi et

al., 2001).

DNA topoisomerases catalyze the unlinking of the DNA strands by making

transient DNA strand breaks and allowing the DNA to rotate around or reverse through

these breaks. DNA topoisomerase I1 inhibitors are the subjects of considerable

phannacologic and clinical investigation. DNA topoisomerase I1 helps to control the

topology of DNA by allowing one strand to pass through the other. This strand passing

also is important in the decatenation of chromoz;omes after DNA replication but before

mitosis. Cancer cells have higher topoisomerase I1 levels. Therefore, inhibition of this

enzyme will lead to a decrease in cell proliferation. Picroliv was found to inhibit the

activity of DNA topoisomerase I1 of Saccharomyces cervisiae mutant cell cultures. Results

of our study clearly showed that Picroliv administration could increase the life span of

ascites tumour bearing animals. Picroliv treatment was also found to reduce the solid

tumour volumes induced by transplantable tumours.

Section 11

Anticarcinogenic activity of Picroliv

Effect of Picroliv on Chemical Carcinogenesis

1. Introduction

Carcinogenesis is a multistage process anti encompasses prolonged accumulation of

injuries at several different biological levels and produce both biochemical and genetic

changes in cells. At each of the levels there is an opportunity for intervention-a chance to

prevent, slow or even halt the gradual change of healthy cells towards malignancy (Peter,

1996). In spite of immense efforts to improve treatment and find cures for advanced

disease, overall mortality rates for most forms of epithelial cancer have not declined in the

past 25 years (Hong and Sporn, 1997). Efforts to prevent the disease would be a more

desirable as well as practical approach for cancer control in contrast to diagnosis and

therapy as modalities of cancer control that are usually costly long-term efforts with only

moderate success. Chemoprevention by pharmacological intervention is an attractive

approach to reduce cancer incidence and mortality.

Liver being the major site for detoxification is the primary target for environmental

or occupational toxic exposure. Because all the blood in the body must pass through it, the

liver is usually accessible to cancer cells travelling in the blood stream. Liver can cleanse

the body of ingested or internally produced poisons, it cannot cleanse it self of cancer.

Primary liver cancer, which starts in the liver, ac:counts for about 2 percent of cancers in

the United States but up to half of all cancers in some under developed countries. Various

carcinogens are associated with primary liver cancer, including aflatoxins, nitrosamines,

vinyl chloride and arsenic. Primary liver cancer is rarely detectable early, when it is most

treatable. The vast majority of primary liver cancer is hepatocellular carcinoma. Liver

cancer is hard to treat, either because of the cancer is too advanced or the liver is too

diseased to permit surgery, at the time of diagnosis. In addition, the liver's complex

network of blood vessels makes surgery difficult. In some patients, radiation and

chemotherapy reduces their tumours to operable size. However, side effects persist and the

patients often succumb to death. Because of these dismal experience in treatment major

efforts should be directed towards the prevention of liver cancer.

Picroliv administration has been shown to be hepatoprotective both in vivo and in

vitro against a variety of toxins (Dwivedi et al., 1990, 1991; Saraswat et al., 1999) and

proved to inhibit the biochemical changes induced by aflatoxin B-1 in rats (Dwivedi et al.,

1993; Rastogi et al., 2000, 2001). Picroliv preconditioning was found to protect liver and

kidney against ischemia reperfusion injury in rats (Singh et al., 2000, Seth et al., 2000).

Recently, Picroliv has been shown to regulate gene expression during hypoxia (Jaddipati et

al., 1999 a; 1999 b). The present section deals with the effect of Picroliv on a)

nitrosodiethylamine-induced liver tumours in rats b) 1,2-dimethylhydrazine-induced

hepatic and renal toxicity in rats. c) 20-methylcholanthrene-induced sarcoma development

in mice d) 7,12-dimethybenz anthrazene- induced papilloma formation in mice.

2. Materials and methods

2.1 Determination of the effect of Picroliv administration on NDEA-induced

hepatocarcinogenesis

The study was performed on 6-8-week-old male Wistar rats weighing 100-120 g.

Rats were randomly divided into four groups (n=XO each). Animals in the group I sewed as

untreated normal rats. Animals in the group I1 to IV were administered with 0.02 %

NDBA, 2.5 mllrat, 5 days weekly for 20 weeks (Narurkar and Narurkar, 1989). Rats in the

group 111 to IV were administered with Picndiv 40 and 200 mg/ kg, respectively

immediately after NDEA administration and continued for 20 weeks. Animals were kept

without NDEA or Picroliv treatment for one week and sacrificed by diethyl ether

anesthesia after an over night fasting. Blood was drawn by cardiac puncture and serum was

separated. Liver was surgically excised, weighed and homogenate prepared was used for

biochemical estimations. A small piece of the liver was fixed in 10 % formalin. The

formalin fixed specimens were embedded in paraffin and sectioned (3-5 pm), sections

from each group were stained with haematoxylin and eosin, and a pathologist analyzed

histological sections.

Serum y-GT activity was assayed by the method described by Szaz (1976). Livery-

GT activity was assayed by the method described by Tate and Meister (1974). GST

activity was determined by the method described by Haibig et a1 (1974). Hepatic GSH was

determined by the method described by Moron et a1 (1979). ALP was assayed by the

method described by King and Armstrong (1980). GPT was assayed by the method of

Bergmeyer and Bernt (1980). Serum LPO was assayed by the method described by Yagi

(1979) and expressed in terms of the amount of miilondialdehyde formed in nmoYml

(Ohkawa et al., 1979). Bilirubin level in the serum was assayed by the method described

by Jendrassik and Jrof (1938). The detailed procedures are given in chapter 11.

2.2 Determination of the effect of Picroliv treatment on DMH-induced toxicity

Male Sprauge Dawley rats (120-150 g) were used for the study. The animals were

allocated randomly to four groups (n = 10 each). Animals in the group 11 - 111 were treated

with Picroliv 40 and 200 mglkg (suspended in 1 ml distilled water p.o), respectively, five

days a week, once daily, for 12 weeks starting from the onset of the experiment. On days

21,28, and 45 of the experiment, rats of group I1 - IV were administered with DMH by i.p.

injection dissolved in a constant volume of PBS (:0.5 rnl, pH 7.4) in doses of 30 mg, 30 mg

and 60 mgfkg body weight respectively (Viswanathan et al., 1998), while animals of group

I received saline alone. At the end of 12 weeks! all the animals were sacrificed after an

overnight fasting by ethylether anesthesia. Blood. was aspirated from the heart and serum

was separated. The liver and kidney were rapidly excised, rinsed in ice cold saline,

weighed and homogenate was prepared for various assays. A small portion of the liver and

kidney were stored in 4 % buffered formalin for histological examination.

Superoxide dismutase (SOD) activity of tissue was determined by NBT reduction

method of Mc Cord and Fridovich (1969). Catalase (CAT) activity was estimated by the

method of Abei (1983) by measuring the rate of decomposition of hydrogenperoxide at

240 nm. In addition, Liver y-GT, GST, GSH, LPO and bilirubin levels were also

determined.

Formalin fixed specimen from each group was embedded in parafh and

sectioned (5 pm). Sections were dewaxed in xylene and rehydrated through graded alcohol

and stained with hematoxylin-eosin (H and E) for routine histopathology. Argyrophilic

Nuclear Organizer Reglon (AgNOR) staining war: carried out by the method of Murray et

al(1989) with modifications as described by Lahshmi et a1 (1993). Detailed procedures are

given in chapter 11. Argyrophilic nuclear organizer regions (AgNOR's) were visualized as

distinct silver positive black dots and clusters. Two hundred nuclei were assessed and the

mean number of dots and clusters per nuclei were calculated separately for each specimen.

2.3 Determination of the effect of Picroliv on sarcoma induced by 20-MC

Male BALBIc mice (6-8 weeks old, 20-2.5 g) were used for the study. Hair was

shaved from the dorsal side of mice. All animals .were administered with a single dose of

20-MC (200 pgi 0. l ml DMSOI mouse) subcutaneously on the dorsal side. This dosage has

been shown to produce sarcoma development in mice by 8-12 weeks (Joy et al., 2000).

Thereafter animals were randomly divided in to four groups (n=15 each). Animals in the

group I was kept without any drug treatment. Animals in the group 11-IV were

administered orally with Pivroliv 50, 100, 200 mgkg body weight, respectively thrice

weekly for 8 weeks. Sarcoma development as well as survival of the animals were noticed

up to 200 days.

2.4 Determination of the effect of Picroliv treatment on papilloma formation initiated

by DMBA and croton oil.

Male BALBlc mice were used for the studies. They were kept as groups of four

animals/cage to reduce fighting and resulting skin aberrations. Aggressive males were

removed and kept separately. The dorsal region (2 cm diameter) of mice were shaved with

a razor at least two days before treatment with DMBA. Only mice which did not show

signs of hair regrowth were used for the experiments. Single dose of DMBA (470

nmoVmouse in 200 p1 acetone) was used in this study (George and Kuttan, 1997). Animals

in group I to V were applied with 10 % croton oil (in 200 p1 acetone) two weeks after

DMBA application. Picroliv was administered either topically dissolved in acetone or

orally suspended in distilled water. Animals were divided as; Group I - DMBA + croton

oil, twice weekly for 6 weeks. Group I1 - DMBA + Picroliv (Imglmouse, topical) 30

minutes before each croton oil application, twice weekly for 6 weeks. Group III - DMBA + Picroliv (5mg/mouse, topical) 30 minutes before each croton oil application, twice weekly

for 6 weeks. Group IV - DMBA + Picroliv (50 mglkg, p.o), 30 minutes before each croton

oil application, twice weekly for 6 weeks. Group V - DMBA + Picroliv (150 mglkg, p.o),

30 minutes before each croton oil application, twice weekly for 6 weeks. Group VI -

DMBA alone. Group VI1 - croton oil alone, twice weekly for 10 weeks. Group VIII -

Picroliv alone (5 mg/mouse (topical), twice weekly for 10 weeks.

In order to check the effect of Picroliv on the initiation of papillomas by DMBA,

another set of animals were used and grouped as: Group I - a single dose of DMBA, two

weeks after animals were treated with 10 % croton oil, twice weekly for 6 weeks. Group I1

- Picroliv (1 rng /mouse. topical), 10 continuous days prior to the application of DMBA

followed by croton oil. Group I11 - Picroliv (5 mg /mouse, topical), 10 continuous days

prior to the application of DMBA followed by croton oil. Group IV - Picroliv (50 m@g,

p.o), 10 continuous days prior to the application of DMBA followed by croton oil. Group

V - Picroliv (150 mgkg, p.o), 10 continuous days prior to the application of DMBA

followed by croton oil. Group VI - Picroliv 5 mglmouse on the shaved area for 10

continous days. Two weeks after croton oil was applied to the skin of animals. Group

VII - a single dose of DMBA. Two weeks after Picroliv (5 mglmouse) was applied to the

skin of animals, twice weekly for 10 weeks.

The animals in all groups were watched for food in take as well as any apparent

toxicity such as weight loss or mortality during the entire period of the study. Skin tumour

formation was recorded weekly, and the tumours greater than 1 mm in diameter were

included in the cumulative total if they persisted for 2 weeks or more. Delays in the onset

of tumours in various groups were recorded.

The values were expressed as means * standard deviations. The results were

analyzed statistically by use of Student's t-test. Values less than 5 % @ ~0 .05 ) were

considered to be indicative of statistical significance.

3. Results

3.1 Effect of Picroliv treatment on NDEA induced hepatocarcinogenesis

All animals In the NDEA administered group (Group-11) had 100 % tumour

incidence by the end of 21" weeks (Plate 5 c). Picroliv administration was found to

significantly inhibit the tumour development in liver, as the tumour incidence was 0 % in

the two groups of animals treated with Picroliv (Table IV.2.1). Increased liver size by

NDEA administration was reduced by Picroliv treatment (Plate 5 e). Liver weight of

NDEA treated animals were raised as compared to normal rats. Picroliv administration

significantly lowered the liver size and liver weight (Table IV.2.2) Increased I-GT, a

marker of hepatocellular carcinoma, in serum as well as in liver was found to be effectively

lowered by the administration of Picroliv, indicating that it could reduce the proliferation

of tunlour cells (Table IV.2.1). Similarly, hepatic GSH, GST and serum LPO values, which

were increased after NDEA treatment, was found to be lowered by the administration of

Picroliv (Table IV.2.2 and IV.2.3). ALP and GPT activity in the serum of NDEA

administered group were raised as compared to that of nonnal value. Picroliv

administration significantly inhibited the rise of ALP and GPT (Table IV.2.3). Similarly,

Table IV. 2.1 Effect of Picroliv administration on tumour incidence, liver weight and y-glutamyl transpeptidase activity of rats administered with NDEA

I ) Normal rats

Group Animal status

11

Tumour incidence

NDEA alone

ID

IV

Liver weight/ 100 g b.w.

NDEA + Picroliv 40 mgi kg

NDEA + Picroliv 200 mg/ kg

I rglutamyl transpeptidase activity I

* p < 0.001, VS group 11. Values are mean i SD (n = 9)

Serum ( m a t 30P C)

Table IV. 2.2 Effect of Picroliv administration on hepatic GSH, GST and serum ALP levels of rats administered with NDEA

Liver (nmoUmin1 mg protein)

Group

I

I1

UI

IV

* p < 0.001, VS group 11. Values are mean 1 SD (n = 9)

Animal status

Normal rats

NDEA alone

NDEA + Picroliv 40 mgi kg

NDEA + Picroliv 200 mg/ kg

GSH (nmoUmg protein)

8.35 0.20

23.67 * 1.53

12.67 i 0.75*

11.10i0.86*

GST (nmoUmin1mg protein)

401 i 11.1

1578 1 130

641 * 76*

633 i 83*

Table IV. 2.3. Effect of Picroliv administration on serum GPT, LPO and bilibubin levels of rats administered with NDEA .

* p < 0.001, VS group 11. Values are mean 5 SI) (n = 9)

Table IV. 2.4. Effect of Picroliv treatment on organ weight and liver 3-glutamyl transpeptidase (7-GT) of rats administered with DMH.

Total bilirubin (mgldl)

16.65 i 0.38

50.89 * 4.41

27.07 * 2.27*

22.83 * 3.77*

LPO (nmoVml)

401.4i 11.1

1578 i 130

641.3 76.4*

633 i 83.5*

Group

I

I1

In

IV

a = p < 0.00 1, d = p < 0.02 as compared to group 11. Values are mean i SD, n = 10.

Group

I

11

III

IV

Animal status

Normal rats

NDEA alone

NDEA + Picrollv 40 mgl kg

NDEA + Picroliv 200 mg 1 kg

GPT (Ulml)

8..35 i 0.20

23 67 i 1.53

12.67 * 0.75*

11.10i0.86*

Animal status

Normal rats

DMH alone

DMH + Plcrollv 40 mg I kg

DMH + P~cro l~v 200 mgi kg

.I-GT

(nmol/min/mg protein)

0.072 * 0.022

0.414 i 0.062

0.226 =+ 0.038 a

0.180 * 0.028 a

Organ weight (g1100g b. w)

Liver

2.74 i 0.18

3 21 * 0.37

3 02 i 0.26

8g 0,16 d

Kidney

0.58 i 0.03

0.62 + 0.16

0.62 i 0.08

0.56 + 0.06

elevated levels of serum LPO and bilirubin of NDEA treated group was also found to be

lowered by the administration of Picroliv (Table IV.2.3).

Histopathological analysis of normal rat liver showed uniformly arranged liver

plates with oval hepatocytes of uniform size (Plate 5 b). NDEA treated rat liver showed

well differenciated hepatocellular carcinoma of trabecular pattern with irregularly formed

cell plates. Scattered masses of necrotic tissues were detected in most of the areas. Nuclei

were enlarged with prominent chromatin and nucleoli (Plate 5 d). Portal areas and hepatic

veins were distorted. Neutrophil infiltration and inflammatory responses were detected.

Sinusoids were compressed. Haemorrhagic blebs were also detected. Hepatocytes

remained normal in the Picroliv treated group (200 mglkg) with,uniform sinusoids (Plate 5

f). However, small emboli of degenerating hepatic cells were detected in some foci.

3.2 Effect of Picroliv treatment in rats administered with DMH.

Administration of DMH resulted in an increase in liver weight as compared to

nonnal rats, which was lowered by Picroliv treatment (Table IV.2.4). Liver y-GT of DMH

administered rats were raised to 0.414 + 0.062 nmoYmg protein as compared to the normal

rat liver value 0.072 ? 0.022 nmollmg protein. Picroliv (40 and 200 mglkg) treatment

reduced the elevated levels of 1-GT to 0.226 + 0.038 and 0.180 rt 0.028 nmoVmg protein,

respectively (Table IV.2.4). Hepatic and renal SOD of DMH treated rats were found to be

5.09 + 2.12 and 3.25 2 0.88 Ulmg protein, respectively. Picroliv treatment (200 mgtkg)

elevated the hepatic and renal SOD values to 9.75 + 1.15 and 6.12 i 1.32 U/mg protein,

respectively (Table IV.2.5). CAT activity of liver and kidney of DMH administered rats

were found to be 31.17 t 14.71 and 18.63 + 7.44 UI mg protein as compared to normal

value of 57.12 * 11.61 and 33.38 2 5.43 UI mg protein, respectively. Picrolv treatment

restored the depleted levels of hepatic and renal CAT levels (Table IV.2.5). Hepatic GST

of DMH (group 11) and Picroliv administered animals (group 111 and IV) were slightly

raised as compared to normal rats. But the depleted renal GST was significantly increased

by Picroliv treatment (Table IV.2.6). Similarly, depleted hepatic GSH of DMH

administered group was significantly reduced by Picroliv treatment (Table 3). DMH

administration significantly increased the levels of malondialdehyde (MDA), an index of

lipid peroxidation, in liver. kidney and serum as compared to normal rats. Hepatc and renal

MDA levels of DMH administered rats was raised to 2.97 + 1.44, 2.78 it 0.60 nmoYmg

Plate 5

Gross MorphoIogy and EIist.jpathsPogy of rat liver

(2) Normal rs; liver ~iiarpho!og;l.

(b) Histology of ii~;rnal rats showing hepa:~cytes of unilam size .md pattern

of arrangemmt (20 X).

(c) Liver of NDEA administered rats showing tumaur nodules.

{d) Histology of NDEA adininiste1,ed rat liver sections showing

anisonucleosis, hyperchromatic nucleus with clubbed chromatin (20 X).

( (5 ) !her of rats treated with Picroliv 200 mgtkg shows absence of tumour

noddes.

(f) Iilsrolcgy of Picroliv (230 mglkg) treated rat liver showing nomai

hep8:ocytes with minimai inflarnn~aiory infiltrate (20 X).

Table 1V. 2. -5. EXecB of kicroliv treatment on glutaBicileS-transferasr! (GST) and glr~tathione (GSH) levels of rats administered with DMIP

Table IV. 2.5. Influence of Picroliv treiatiz.:nt on superoxide dismutase and catalase activity of rats administered with D m .

-

i Supreoxide dismutase 5JImg protein)

I , Liver Kidney I -

. . - .. . .- - -

Group Animal stqtus , GST (amoUmin/mg protein) t-- i Liver IGdney Liver Kidney

Catalase (Ulmg protein)

-- -.

-. -

; Jornlal rats I

I i 5 9 4 i 5 8 459 * 23 i 8.22 + 0.67 i

I1 DMF[ alone

Elver

6.25 * 0.67

Kidney

33.38i5.43

18.63 f 7.44

33.12i 5.04 a

31.67 i 3 . 3 a

t-!xmai rats I

8.56i0.96 5.76i0.33 . 57.12i 11.61 i I

I II

I DMH alone ; 5.09 i 2.12 3.25 i 0.88 1 31.17i 14.71

I r

I i !

I_ I 1 .- -L -. -. I - n = 10

L- s. = p 0.001, b = p ; O.CO5, c = ;j 0.01 ns compared to group 11. Values are mean i SD,

I f-

52.11 i 26.17 DI

IV / 9-75 + 1.15 ' 1 6.12 i 1.32 a 62.55 i 15.22 a

' 6.44 i 1.90 5.43 i z.04 a 4Limdkg i

DMH t Picroliv ' 208 mg/ kg

I

5.81 i 0.64

5.77 i 0.72

6.50i1.27

S : = p < 0.01, d = p -: 0.02 as com2ared t3 group !I. Values are mean rt SD, n = 10.

6.82 * 1.71

8.76 i 1.97

8.94 i 1.55 d

1 6911 116 I 368 i 56

m DMH + Picroliv 40 mg/ kg

I IV DM1 I t Picroliv 200 mg/ kg

6 5 6 i 8 3 1 4 1 1 i 8 7

630 i 59 443 i 42

I !

I I .---I_._. L-- - i

protein, respectively. Picroliv treatment (200 mgkg) lowered the MDA levels to 1.44 &

0.61 and 1.54 * 0.71 nmoUmg protein, respectively. Increased serum MDA value (3.55 i

0.91 nmoUml) of DMH administered rats was reduced (2.25 i 0.47 and 1.87 i 0.60

nmollml) by Picroliv 40 and 200 mgkg, respectively (Table IV.2.7). Elevated bilirubin

level of DMH administered animals was reduced by Picrolv treatment (Table IV.2.7).

H and E staining of DMH administered rat liver shows the presence of hepatic cell

necrosis (Plate 6 a) and nodular regeneration. Some areas of the liver section revealed

coalescent nodular areas that distort or replace the normal hepatic structures. At the

surroundings of the nodules, there was cystic hyperplasia of the bile ducts with

inflammation. Localized neutrophil infiltration was noticed in some areas. However,

neoplastic transformation was not detected in liver sections. Picroliv treated rat liver

resembles to that of normal in most areas (Plate 6 b) except for the presence of a few

degenerating liver cells. AgNOR's dot and cluster of DMH administered rats were

increased (Plate 6 c) as compared to normal rats. AgNOR dots and clusters of normal rats

were found to be 1.21 and 0.48, which were raised to 2.80 and 1.22, respectively by DMH

administration. AgNOR dots and clusters were reduced by Picroliv treatment (Plate 6 d).

Picrolv treatment 40 and 200 mgkg reduced the dot value to 1.44 and 1.48 and cluster

value to 0.89 and 0.59, respectively. Three injections of DMH as used in the present study

was found to be insufficient to produce histological changes in kidney (Plate 6 e,f) and

colon.

3.3. Effect of Picroliv on the development of sarcomas induced by 20-MC

Picroliv administration inhibited the sarcoma development induced by 20-MC in a

dose dependent manner (Table IV.2.8) and increased the life span of sarcoma bearing

mice. Sarcoma size was small in the group of animals treated with Picroliv (Plate 4 f)

Administration of Picroliv (50, 100 and 200 mgfkg) inhibited the sarcoma development by

13, 47 and 53 O/o as estimated on 200'~ day after 20-MC administration. There was also a

delay in the development of sarcomas in the group of animals treated with Picroliv 150

mglkg (9 weeks on test) and 200 mgkg (8 weeks on test) as compared to the control group

of animals in wh~ch the first sarcoma was noticed 6 weeks on test. Control animals started

dying of tumour burden (Plate 4 e) 76 days after 20-MC administration and all animals

Plate 6

Histology of liver and kidney of rats

(a) Liver section of rat administered with DMH showing necrosis

(b) Picroliv (200 nlg/kg) treated rat liver cells showing r~onnal size and

arangement of hcpatocytes.

(c) AgNOR staining of DM11 administered rat liver shwiving increased

&NOR dots and clusters (100 Xi;).

(d) Picraliv treated rat liver cells showiag AgNOR dots similar to that of

normal hepatocyies (100 X).

(el Kidney hisiologj. d DMH administered rats (40 X ) .

!'fi Kidney histology of Picroliv (200 mg/kg) treated rsis (LLO X;.

Plate No:6

Table IV. 2.7. Effect of Picroliv treatment on lipidperoxidation and serum bilirubin levels of rats administered with DMH

a = p < 0.001, b = p < 0.005, d = p < 0.02 as compared to group 11. Values are meanISD,n= 10.

Group

I

I1

m

N

Table 1V.2.8. Effect of Picroliv administration on 20-MC induced sarcoma development in mice.

Animal status

Normal rats

DMH alone

DMH + P1croliv 40 mg/ kg

DMH + Picrol~v 200 mg/ kg

Days

60

80

100

120

140

160

180

200

Total bilirubin (mg1100 ml)

0.44* 0.08

1.6 8 i 0.52

0.88 * 0.32

0.66 * 0.23 a

Lipid peroxidation

Number of animals developed sarcomas

Control

1/15

4:15

8i15

11/15

13115

15/15

15/15

15'15

Serum (nmoVml)

1.69 i 0.25

3.55 0.91

2.25 i 0.47

1.87 i 0.60 a

Liver (nmoVmg protein)

1.07 * 0.35

2.97 i 1.44

* 0.80

1.4 * 0.61 d

Kidney (nmoUmg protein)

1.30 i 0.13

2.78 i 0.60

1.88 * 0.41

1.54 i 0.71

Picroliv 50 mgkg

1/15

3/15

5/15

8/15

10115

13115

13115

13115

Picroliv 100 mgkg

0115

2/15

3/15

5/15

6/15

6/15

7/15

8/15

Picroliv 200mgkg

1/15

1/15

1/15

3/15

4/15

4/15

5/15

7/15

were dead by the 170'~ day, while 20.60 and 66 % animals survived in the Picroliv treated

group 50, 100 and 200 mgkg b.w, respectively (Table IV.2.9).

3.4. Effect of Picroliv on papilloma formation induced by DMBA and croton oil

Topical application of Picroliv prior to croton oil administration in DMBA-initiated

mice resulted in a significant protection against skin tumour promotion in a dose dependent

manner. Picroliv administration substantially lowered the percent of mice with tumours

and decreased the total number of tumours per mice (Table IV.2.10). Time of the

appearance of the first tumour in Picroliv treated group 50 mgkg was delayed by 2 weeks

and there was an 8 weeks delay in tumour development in animals treated with Picroliv

150 mgtkg. At the termination of the experiment, at 20 weeks, all the animals in the control

group developed tumours (Plate 4 g), while only 50 and 25 % animals in the Picroliv

painted group and 55 and 50 % animals in the Picroliv orally treated group exhibited skin

neoplasms. There was also a decrease in the number of papillomas per tumour bearing

mouse in animals treated with varying dose of Picroliv (Table IV.2.10, (Plate 4 h).

Picroliv administration prior to DMBA application showed an inhibition in

papilloma development in mice, indicating that Picroliv had an effect on the tumour

initation process (Table IV.2.11). These inhibitory effects were also dependent on the dose

of Picroliv. Compared to the control animals, in which the first tumour appeared at 7

weeks after DMBA application, treatment with 50 and 150 mglkg body weight of Picroliv

resulted in a 2 and 4 weeks delay on the onset of first tumour, respectively. Topical

application of Picroliv was found to be in effective in delaying the tumour appearance.

Picroliv administration was found to be effective in inhibiting the number of papillomas in

papilloma bearing mice (Table IV.2.11).

Administration of DMBA (470 nmol/mouse) did not produce any tumours

suggesting that this dose level was ineffective to elicit carcinogenic potential without

further promotion. Croton oil and Picroliv alone did not produce any tumours suggesting

that they are not carcinogenic. Moreover, animals topically treated with Picroliv (5

mglmouse) and promoted with croton oil were also devoid of tumours suggesting that

Picroliv it self is not an initiator. Similarly, animals initiated with DMBA and promoted

with Picroliv (5 mglmouse) were devoid of any tumours up to the termination of the

Table IV.2.9. Effect of Picroliv treatment on the survival of animals administered with 20-MC.

Table IV.2.10. Effect of Picroliv administration on papilloma induction initiated by DMBA and croton oil.

Days

60

80

100

120

140

160

180

200

I ( DMBA + croton oil I 717

Number of animals survived

Group

Control

15/15

14/15

12/15

10115

3115

2115

0115

011 5

Animal status

I1

Picroliv 50 mgkg

15/15

15/15

13115

10115

7/15

711 5

411 5

311 5

Number of mice developed papilloma

by 20 th weeks

111

DMBA + croton oil +Picroliv

1 mg/mouse (topical)

IV

VII ( Croton 011 alone ( 018

Picroliv I00 mgkg

15/15

15/15

15/15

14/15

12/15

10115

10115

911 5

418

DMBA + croton oil +Picroliv 5 mg/ mouse(topica1)

V

VI

Picroliv 2OOmgkg

15/15

15/15

14/15

14/15

12/15

12/15

1111 5

1 011 5

4/10

DMBA + croton oil +Picroliv 50 mg/

kg(ora1)

519

DMBA + croton oil +P~crollv 150 mglkg

(oral)

DMBA alone

VII

Number of papillomas per

tumour bearing mice

418

017

Percentage redution in papillomas per

tumour bearing mice

* P < 0.001. * P < 0.005 Vs group I

Plcrol~v alone(5mgi mouse, top~cal)

019

experiment at 20 weeks, suggesting that Picroliv is not a tumour promoter (Table

IV.2.11).

4. Discussion

Cancer development is a multifactorial process (Cohen and Ellwein, 1991). After a

carcinogen has been taken up, and transported to its target (presumably DNA), the

carcinogenic process may be further modified by a number of dynamic processes inherent

to the host or its tissues. Such properties as DNA repair processes; cellular proliferative or

trancriptional status enhances or suppresses carcinogenicity by affecting the fixation and

maintenance of a carcinogenic lesion. Recently, there has been growing interest on the role

of free radicals and lipid peroxidation at the tissue level as a cause of cancer. There is

evidence indicating that generation of active oxygen species and formation of reactive

products may be involved in various carcinogenic processes. There are powerful

antioxidant defence mechanisms against the toxic effects of active oxygen species in the

body. Among them, superoxide dismutase and catalase are the important enzymes.

Treatment with carcinogens or tumour promoters usually decreases levels of superoxide

dismutase and catalase (Slaga, 1995). A decline in these enzymes may facilitate the

initiation of oxidative processes, which would lead to the elevation of reactive oxygen

species and consequently may account for increases in levels of oxidized DNA bases,

credited for mutagenesis and carcinogenesis.

The results presented in this study indicate the protective action of Picroliv, iridoid

glycoside mixture, isolated from Picrorhiza kurroa against chemically induced tumours.

NDEA and methylcholanthrene are ubiquitous environmental carcinogens and their

carcinogenicity has already been demonstrated in several animal species. In addition, they

could be formed endogenously in the body. The limited treatment options and poor

treatment success HCC a leading cause of death in developing countries. Treating patients

with HCC is a disappointing experience given the lack of effective tumoricidal agents. In

this context, the results obtained in this study are remarkable because Picroliv treatment

could significantly inhibit hepatocarcinogenesis induced by NDEA. This was reflected in

the absence of tumours in Picroliv treated groups and significant changes in marker

enzymes such as y-GT, GST, ALP, GPT and other liver injury markers such asbilimbin.

Picroliv when given orally was found to significantly reduce the development of sarcoma

Table IV.2.11. Effect of Picroliv administration on the initiation of papillomas induced by DMBA.

* P < 0.005 Vs group I

Percentage redution in papillomas per

tumour bearing mice

9.8

25.4

29.4

66.6

Number of papillomas per

tumour bearingmice

5.1 i 1.8

4.6 i 1.5

3.8 i 1.3

3 . 6 i 1.3

1.7 0.8 *

Number of mice developed papillomas

by 20 th weeks

6/6

819

519

619

418

018

019

Group

I

I1

111

IV

V

VI

VII

Animal status

DMBA + croton oil

DMBA 4- croton oil +Picroliv

I mglmouse (topical)

DMBA + croton oil +Picroliv 5 mg/ n~ouse(topical)

DMBA + croton oil +Picroliv 50 mg/

kg(oral)

DMBA + croton oil +Picroliv 150 mgkg

(oral)

Picroliv (5 mglmouse topical) + croton oil

DMBA + Picroliv (5 n~gtrnouse, topical)

and accompanied death produced by the injection of 20-MC. Picroliv administration was

also found to reduce the papilloma formation during two-stage carcinogenesis model

induced by DMBA as initiator and croton oil as promoter. In this model, Picroliv was

found to inhibit the papilloma formation when applied topically as well as given orally. In

fact, oral administration was found more effective in reducing the number of papilloma

bearing animals and the number of papillomas per mouse. More over, Picroliv treatment

(oral and topical) prior to DMBA administration could inhibit the initiation produced by

the carcinogen. However, activity was more significant when the administration was

continued during the promotion stage induced by croton oil.

DMH is a potent necrogenic hepatocarcinogen (Ying et a]., 1979; Hawks et al.,

1974) that alkylates hepatocellular DNA leading to carcinogenesis (Swenberg et a]., 1979).

DMH is metabolized rapidly by the liver and it induces zonal necrosis and oxidative stress

(Hayes et al., 1987). Necrosis can promote hepatocarcinogenesis by enhancing growth of

initiated hepatocytes resistant to toxicity (Farber and Sharma 1987; Ying and Shanna,

1981). DMH administration has been shown to induce hepatocellular carcinoma and

angiosarcoma in mice (St. Clair et al., 1990). DMH is also a powerful colon carcinogen

(Corasanti et al., 1982; Weed et al., 1985) which induces colorectal tumours in

experimental animals (Ohno et al., 2001).

Our study shows that Picroliv treatment completely prevented DMH-induced liver

necrosis. Picroliv treatment was also found to reduce the elevated levels of y-GT, a marker

enzyme of preneoplatic lesions. WHO (1990) has recognised elevated levels of y-GT in

liver cells as a valid marker of preneoplasia in short-term animal experimentation in

animals such as rats. In our study, DMH administration showed that-a more than five-fold

increase in liver y-GT levels indicating associated neoplastic changes. Picroliv treatment

resulted in a significant ( p <0.001) decline in y-GT levels. Similarly, Picroliv treatment

also reduced the elevated number of AgNOR dots and clusters induced by DMH

administration. It has been suggested that AgNORs represent ribosomal DNA transcription

activity or transcription potential (Eagan and Crocer, 1992; Mourad et al., 1992) and hence

an active reflection of proliferative activity.

We have also assessed the potential of Picroliv by studying its effect on lipid

peroxidation, which was measured in terms of MDA, a stable metabolite of the free

radical-mediated lipid peroxidation cascade. MDA levels rose in the DMH treated group

indicating increasing degree of oxidant damage. Picroliv treatment rescued the hepatic and

renal tissues against DMH-induced lipid peroxidation, a free radical initiated chain

oxidation of unsaturated lipids, involved in cell and tissue damage. Lipid peroxidation

leads to the degradation of lipid membrane. DMH administration resulted in decrease in

hepatic and renal SOD and CAT, the primary defence against oxidation damage of tissues.

SOD plays a role in scavenging of superoxide anion, which is the initial free radical among

the oxygen radicals. CAT prevents oxidative hazard by catalyzing the formation of water

and oxygen from hydrogen peroxide. Increased exposure to radicals or from impaired

efficiency of these protective enzymes lead to diseases including cancer. In this study, we

have found that, Picroliv administration significantly increased the levels of SOD and CAT

as compared to DMH treated animals. Elevated bilirubin, an index of liver damage, by

DMH administration was significantly reduced by Picroliv treatment. Picroliv treated rats

exhibited higher GSH content than DMH administered group, indicating that Picroliv

helped in replenishing the GSH pool. Picroliv feeding has been shown to induce GST, an

important enzyme involved in detoxification of toxic xenobiotics (Rastogi et al., 1995).

Our study showed that Picroliv treatment could significantly increase the depleted renal

GST observed in DMH administered animals. Similar protection of biochemical

parameters such as SOD, CAT, GSH, GST and inhibition of the elevation of y-GT and

lipid peroxidation was noticed by Picroliv treatment in aflatoxin B-1 administered

rats (Rastogi et al., 2001). The results obtained in the study clearly showed that Picroliv

could inhibit the Carcinogenesis induced by NDEA and 20-MC. Picroliv treatment was

also found to inhibit the tumour development in initiated by DMBA and promoted with

croton oil. Picroliv treatment ameliorated the hepatic and renal damage induced by DMH.

Section I11 ,

Radioprotective and Chemoprotective

activity of Picroliv

Radioprotective and Chemoprotective activity of Picroliv

1. Introduction

Systematic cancer chemotherapy agents control turnour growth by interfering with

the proliferation of cancer cells. Because cell replication is characteristic of normal cells as

well as cancer cells, chemotherapeutic agents often have undesirable effects on normal

cells, particularly those with a rapid turnover rates. Mylosuppression has ben found one of

the major drawbacks in cancer chemotherapy. Administration of antineoplastic agents such

as cyclophosphan~ide (CYP) and cisplatin leads to immunosuppression, which at times

leads to life threatening situations. Radiation exposure induces leukopenia as well as

formation of highly reactive free radicals. Use of immunopotentiating agents along with

other modalities of cancer treatment to restore the immunologic system, has been

suggested. Immunostimulants such as Bacilles Calamette Guerin (BCG) levamisole

(Mihich , 1982) and cytokines (Lieschke and Burgess, 1992) along with chemotherapy has

been shown to reduce myelosuppression and leukopenia induced by chemotherapeutic

agents. Rasayana, a non-toxic herbal preparation has been shown to improve the

haemopoetic cells in mice treated with CYP (Praveenkumaret al., 1994) and radiation

(Praveenkumar et al., 1996). Rasayana administration accelerated myeloid recovery in

patients treated with a combination of radiotherapy and chemotherapy (Joseph et al.,

1999). Herbal drugs offer distinct advantages over currently available immunostimulating

agents as they are effective as given orally, have a low preparation cost and most often

non-toxic.

Picroliv has been documented to possess immunostimulant activity (Puri et a].,

1992). The present study was designed to assess the effect of Picroliv treatment in mice

administered with cyclophosohamide and irradiated with Co 60.

2. Materials and methods

2.1. Determination of the effect of Picroliv on mice irradiated with Cobalt 60.

Male BALBIc mice (25 g) were divided in to three groups. Animals of group-I

( n = 6 ) was kept as normal animals. Whole body irradiation (6 Gylmice, 1GyImin) was

given to animals in group I1 and I11 ( n = 18) using Cobalt-60 Teletherapy unit (Theratron

780 Canada). For this, animals were kept in specially constructed restraining box with a

capacity of holding 10 mice. Animals in the group-111 were treated with Picroliv 200

mgkg. The Picroliv treatment started 10 days before radiation administration and

continued for one month after Co 60 irradiation on every alternate day. Six animals from

group-I1 and 111 were sacrificed by cervical dislocation on day 2 and 8 after radiation to

assess bone marrow cellularity, organ weight and biochemical parameters. Blood was

collected from the tail vein on every six days and peripheral leukocyte count and

hemoglobin levels were recorded. Animals in all groups were sacrificed 30' day after

irradiation and biochemical parameters were assessed

Bone marrow cellularity was determined by the method described by Sredni et a1

(1992). Briefly, the femurs were surgically removed, and the bone marrow was flushed out

of the medullary cavity and collected in PBS containing 2 % goat serum. The number of

cells were counted using a haemocytometer and expressed as total live cells per femur.

Hemoglobin was estimated by Drabkin's method using a kit. Total leucocyte was counted

using a haemocytometer. Serum and tissue lipid peroxidation (Yagi, 1984) were

determined and expressed in terms of malondialdehyde formed. GSH was estimated by

the method described by Moron et a1 (1979).

2.2. Determination of the effect of Picroliv on cyclophosphamide-induced toxicity

Male BALBIc mice (25 g) were divided in to three groups. Group-I (n = 6 ) was

kept as normal animals. Animals in group I1 and 111 (n = 18) were administered with CYP

50 mgkg for 10 continuous days. This dosage has been shown to produce severe

mylosuppression in mice (Praveenkumar et al., 1994). Animals in the group-111 were

treated with Picroliv 200 mgkg. The Picroliv treatment started 10 days before the

administration of CYP and continued for one month after the first dose of CYP on every

alternate day. Blood was collected from the tail vein on every six days to determine WBC

and hemoglobin levels. Six animals from group-I1 and 111 were sacrificed by cervical

dislocation on day 2 and 8 after CYP administration to assess bone marrow cellularity,

organ weight and biochemical parameters. The remaining animals in all groups were

sacrificed 3oth day after CYP administration.

3. Results

3.1 Effect of Picroliv on mice irradiated with Cobalt 60

Radiation exposure was found to increase both the serum and liver lipid

peroxidation levels (Table. IV.3.1). Lipid peroxidation levels in serum of normal mice was

found to be 2.9 -t 0.2 nmollml and that of liver was 1.2 + 0.3 nmoWmg protein. Radiation

exposure increased the serum lipid peroxidation value to 5.8 k 0.6 nmol/ml and the liver

value increased to 2.5 ? 0.8 nmollmg protein. Picroliv treatment significantly reduced the

elevated values (Table IV.3.1). Radiation exposure resulted in the reduction of bone

marrow cellularity. Significant increase in bone marrow cellularity was noticed during

Picroliv treatment (Table IV.3.2). There was also a sharp decline in hepatic GSH levels of

mice irradiated with Co 60. Picroliv treatment augmented the recovery of depleted GSH

(Table IV.3.2). Picroliv treatment not only prevented the decline of leukocytes and

increased the leukocyte levels during treatment as compared to untreated mice (Fig

IV.3.1). Hemoglobin levels were declined during radiation administration and Picroliv

treatment restored it to that of normal levels by 30" day (Fig IV.3.2). There was no

significant change in body weight and organ weight of animals treated with Picroliv and

radiation as compared to normal animals (Table IV.3.3 and 3.4).

3.2 Effect of Picroiiv on cyclophosphamide-induced toxicity

Picroliv treatment was found to increase the leukocyte count of mice treated with

CYP (Fig IV.3.3). In the control group (CYP alone) the total number of WBC dropped to

6819,1188 cellslmm ' on 2nd day and the value reached 6708 * 1256,7260 k 731,8180 + 971,8290 k 688 and 8490 k 886 cellslmm on day 8, 14,20,26 and 30 day, respectively

after CYP administration. Significant increase in WBC count was noticed in Picroliv

treated animals on day 14 '~, 20Ih, 26Ih and 30" as compared to CYP administered animals

(Fig IV.3.3). However. haemoglobin content was not significantly altered by Picroliv

treatment (Fig IV.3.4). Haemoglobin content on day 30Ih after CYP administration was

found to be 13.12 +- 1.22 and 14.62 + 0.89 g/100 ml in animals administered with CYP

alone and Picroliv, respectively. Bone marrow cellularity in normal animals was found to

be 14.46 k 1.73 x 10 6. CYP administration resulted in the suppression of bone marrow

cellularity and the value reached 4.90 k 0.81 and 9.00 + 2.59 x 10 on day 8" and 30"

after CYP administration, respectively. Picroliv treatment significantly accelerated the

Fig IV.3.1. Effect of Picroliv treatment on total leukocyte count of mice irradiated with Cobalt 60 (6Gylmouse)

14000 -

12000 -

10000 -

"E 8000 - 3 s

% 6000 - 0 z

4000 -

2000 - -A- Radiation alone

0. 2 8 14 20 26 30

Days

a = p < 0.001, b = p < 0.005, c = p < 0.01, d = p < 0.02 as compared to Radiation alone

Fig IV.3.2. Effect of Picroliv treatment on haemoglobin content of mice irradiated

with Cobalt 60 (6Gylmouse)

-0- Normal mice -A- Radiation alone -W- Radiation + Picroliv 200mglkg

0 -1 I

2 8 14 20 26 30 Days

Fig IV.3.'3. Effect of Picroliv treatment on total leukocyte count of mice administered with cyclophosphamide

-A- CYP alone 4000 -

2000 -

0 I

2 8 14 20 2 6 30 ' > -

Days

b = p < 0.005, c = p < 0.01, d = p < 0.02 as compared to CYP alone

Fig IV.3.4. Effect of Picroliv treatment on haemoglobin content of mice administered with cyclophsphamide

0 . 2

1

8 14 20 26 30

Days

Table IV.3.1. Effect of Picroliv treatment on serum and liver lipid peroxidation levels of mice irradiated with Cobalt 60 (6 Gylmouse)

c = p < 0.01, d = p < 0.02 as compared to group 11. Values are mean * SD, n = 6 . ND = not determined

Table IV.3.2. Effect of Picroliv treatment on bone marrow cellularity and liver glutathione (GSH) of mice irradiated with Cobalt 60 (6 Gylmouse)

b = p < 0.005, c = p < 0.01 as compared to group 11. Values are mean * SD, n = 6 . ND =not determined

Table IV.3.3. Effect of Picroliv treatment on body weight of mice irradiated with Cobalt 60 (6 Gylmouse)

Table IV.3.4. Effect of Picroliv treatment on the organ weights of mice irradiated with Cobalta(6 Gylmouse)

Group

I

I1

-

ND = not determined

Animal status

No-almlce

Radlatlon alone

Radlatlon+ P~crollv

200 mg /kg

Group

I

m

Body weight (g) on various days after Co 60 irradiation

Animal status

Normalmice

Radiation alone

Radiation+ picro]iv

200 rng k g

2"d day

2 0 . 7 ~ 1 . 1

19.151.2

20.150.8

Organ weights on various days after Co 60 irradiation

8" day

22.451.7

18.651.1

17.651.3

Liver

14" day

23.9i0.2

21.6k2.2

21.5*2.1

30" day

1.22+0.21

1.40*0.26

2"' day

ND

1.29*0.ll

Kidney

8" day

ND

1.66iO.321.5610.221.44i0.32

1.38i0.18

2" day

ND

0.3i0.03

0.3+0.02

Spleen

30" day

24.1i0.9

22.6i1.1

22.1*1.4

20nd day

22.450.6

20.1i1.8

22.1i1.6

2d day

ND

0.1+0.02

0.08+0.020.07~0.02

26" day

25.1i0.8

23.1i0.9

24.1i1.3

8" day

ND

0.410.03

0.3*0.03

30h day

0.3 + 0.06

0.4 10.04

0.3i0.04

day

ND

0.08+0.01

30m day

0.07+0.02

0.09+0.01

0.07*0.01

recovery of bone marrow cellularity (Table IV.3.5). Depleted hepatic GSH of CYP

administered animals were significantly increased by Picroliv' treatment cable IV.3.5).

Picroliv treatment was found to be ineffective in reducing serum and liver lipid

peroxidation on day 2 and 8 after CYP administration. However, the levels were

significantly @ < 0.02) reduced by Picroliv treatment on 3oh day after CYP administration

(Table IV.3.6).

4. Discussion Development of strategies capable of protecting normal host tissues from the lethal

actions of ionizing radiation without compromising antitumour effects may permit

improvement of the therapeutic index of this important modality in the treatment of human

malignancies. The ability of ionizing radiation to kill tumour cells through induction of

DNA damage makes this an important modality in the therapeutic weapon against cancer

in humans. Unfortunately, normal human tissues are not immune to the damaging effects

of ionizing radiation; instead, they represent a target, which limits both the total amount

and rate of administration of radiation that can be safely administered. In general, rapidly

dividing tissues, such as cells of the hematopoietic system and gastrointestinal tract are the

most vulnerable to radiation-induced injury. Amelioration of the toxicity of ionizing

radiation toward these normal tissues might pennit higher radiation doses to be

administered, leading in turn to a net improvement in therapeutic index and antitumour

efficacy. Ionizing radiation exerts its biological effects either directly, through action

against the critical targets such as DNA or indirectly, through infliction of damage by free

radical generated from the radiolysis of water. While the later process has been considered

a major contributor to induction of damage in biological systems, the magnitude of its

effect is influenced substantially by the concentration of free radical scavengers. Human

body has inherent rnechanisms to reduce free radical injury by enzymatic and non-

enzymatic means. At certain times, these natural protective mechanisms may not be

sufficient. Supplementation of non-toxic antioxidants may have a beneficial role in these

conditions

Radiation-induced free radicals produce peroxidation of lipids, leading to structural

and functional damage to cellular membranes. The natural antioxidant systems of the body,

consisting of GSH and related enzymes are believed to be an important part of cellular

Table IV.3.5. Effect of Picroliv treatment on bone marrow cellularity and liver glutathione (GSH) content of mice administered with cyclophosphamide

GSH (nmoUmg protem)

b = p < 0.005, d = p < 0.02 as compared to group 11. Values are mean 5 SD, n = 6. ND =not determined

Table IV.3.6. Effect of Picroliv treatment on serum and liver lipid peroxidation levels of mice administeredwith cyclophosphamide.

d = p < 0.02 as compared to group 11. Values are mean i SD, n = 6. ND = not determined

Group

I

m

Animal status

Nom~al mice

CYPalone

CYP+Plcroliv 200 mg /kg

Lipid peroxidation

Serum (nmoVml) Liver (nmoUmg protein)

30bday

1.87 i 0.29

2.83 i 0.49

1.93+0.37d

Pd day

ND

3.66 10.51

2.86-tO.40

30" day

1.42i0.31

2.7410.84

1.40+0.38d

81h day

ND

3.15 * 0.86

2.6410.39

2nd day

ND

3.80 1 1.23

2.60i0.74

-

@day

ND

3.24 0.76

2.22*0.69

defence against a large array of injurious agents. Under normal conditions, the inherent

defence system, including GSH and related antioxidant enzymes, protects against oxidative

damage. In the intact and healthy cells, GSH and related enzymes are restored immediately

after each interaction. However, in the irradiated animals the normal synthesislrepair will

be disrupted due to damage to DNA and membranes. Oxidative stress due to the radiation-

induced free radicals can cause a dramatic fall in the hepatic GSH and related enzymes

Consequently, restoration will be delayed until. the cells recovered. There was a drastic

reduction in hepatic GSH after irradiation. Picroliv treatment not only prevented the

depletion of hepatic GSH but a1 so augmented the restoration of GSH. The basic effect of

radiation on cellular membranes is believed to be the peroxidation of membrane lipids.

This leads to permeability changes and secondary alterations in membrane proteins. In the

present study, lipid peroxide levels were found to be increased in serum as well as in liver

of irradiated mice and animals administered with CYP, suggesting the associated cellular

damage. The significant reductions in the lipid peroxidation by Picroliv treatment clearly

demonstrate that Picroliv protect the membranes against radiation and CYP induced

oxidative damage. Picroliv treatment was found to increase the bone marrow cellularity

and WBC count of mice administered with radiation and cyclophosphamide, indicating

that Picroliv may reduce the damage to the hemtopoitic system induced by radiation and

CYP.

One of the characteristics that distinguish cancer chemotherapeutic agents from

most other drugs is the frequency and severity of anticipated side effects at usual

therapeutic doses. Tissue injury, especially cells of immune system, represent one of the

factors limiting the administration of chemotherapeutic drugs in cancer treatment. Use of

immunomodulators in cancer therapy is gaining great momentum in recent years.

Immunomodulators can augment the immune response leading to increased tumour cell

kill (Waksel, 1978) and reduce leukocytopenia induced by the cytoreductive therapy used

in the management of cancer (Hersh, 1982).Our study, clearly demonstrate that Picroliv

has the capacity to accelerate hematopoitic recovery and reduce mylosuppression induced

by radiation and CYP