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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