Molecular and Cellular Biochemistry 238: 1–9, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

1

Modulation of chromatin organization by RH-3, a

preparation of Hippophae rhamnoides, a possible
role in radioprotection
I. Prem Kumar, Samanta Namita and H.C. Goel
Department of Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, INMAS, Delhi, India

Received 18 June 2001; accepted 3 December 2001

Abstract
The present study was aimed to understand the mode of action of alcoholic extract of whole berries of Hippophae rhamnoides
(RH-3) which has already been reported to render more than 80 % protection against radiation induced mortality in mice. Direct
and indirect antioxidant action (free radical scavenging and metal chelating potential) were assayed using 2-deoxy ribose deg-
radation and 2,2′-bipiridyl assays. Effect of RH-3 on radiation and chemical oxidant mediated DNA damage was evaluated
using single cell gel electrophoresis (Comet assay) and alkaline halo assay. Ability of RH-3 to bind with calf thymus DNA was
assayed through change in melting temperature (Tm) while toxicity was assayed in thymocytes by trypan blue exclusion.
RH-3 inhibited 2-deoxy ribose degradation in a dose dependent manner (IC 50 ~ 500 µg/ml). 2,2′-bipiridyl assay revealed
the inability of RH-3 to chelate Fe2+ ions. RH-3 inhibited radiation and tertiary butyl hydroperoxide induced DNA strand breaks
in a dose dependent manner and at concentrations of 100 and 120 µg/ml the length of comet tail was considerably reduced and
became almost similar to that of untreated control. RH-3 at a concentration of 120 µg/ml or more induced a strong compaction
of chromatin as was evident from lack of tail and appearance of intensely stained circular bodies. This made the nuclei resist-
ant even to a radiation dose of 1000 Gy. The compaction of chromatin was not reversed even by relaxation buffer indicating
that salt concentration had no role in RH-3 induced chromatin compaction. Alkaline halo assay also corroborated the results of
comet assay. Lower DNA-RH-3 concentrations (1:0.5 and 1:1) induced a shift of Tm towards left by 2 and 5°C respectively;
however higher concentrations (1:8 and 1:16) shifted the Tm towards right increasing it by 10 and 21°C correspondingly.
RH-3, evinced only a mild free radical scavenging activity at concentrations used in the present study, therefore its ability to
protect DNA could mainly be attributed to direct modulation of chromatin organization. Further work to unravel these facts
would be necessary. (Mol Cell Biochem 238: 1–9, 2002)

Key words: Hippophae rhamnoides, chromatin condensation, comet assay, melting temperature, DNA damage, radioprotection

Introduction
Low LET gamma radiation cause damage to the cellular sys-
tem mainly by two different modes: (a) direct energy depo-
sition and degradation of crucial cellular macromolecules like
DNA [1, 2] and (b) generation of reactive oxygen species.
Among the later, hydroxyl radicals are most reactive and are
mainly responsible for the damage. Besides radiation, pol-
lutants and many diseased conditions including cancer also
generate such reactive species [3]. The spectrum of the dam-
age inflicted by the free radical to the cellular machinery
includes lipid peroxidation, oxidation of proteins, base modi-
fications, adduct formation and strand breaks in DNA and
these have been implicated in radiation-induced apoptosis
and cell death [4, 5]. The damage to the cellular DNA since
has been found to be one of the determining factor in radia-
tion induced cell death, efforts are continuing to identify and
exploit agents which can protect DNA against radiation dam-

Address for offprints: H.C. Goel, Department of Radiation Biology, INMAS, Delhi-110 054, India (E-mail: radbiol@nda.vsnl.net.in)
2
age through mechanisms like free radical scavenging [6],
modulation of chromatin structure, repair enhancement etc.
[7, 8]. The inability of the existing agents to fulfil the crite-
ria of an ideal radioprotector, and the inherent toxicity mani-
fested at the useful concentrations has necessitated the search
for better agents [9, 10]. In this context presently the atten-
tion has shifted from synthetic molecules to natural plant
products, which have already been extensively exploited in
traditional medicine systems like Ayurveda, Tibetan and Chi-
nese systems of medicine etc. A number of plant products in
crude form or their isolated constituents, like flavonoids etc.
have been reported to render radioprotection both in vitro and
in vivo conditions [11–14]. H. rhamnoides L. (F. Elaegnaceae),
commonly known as seabuckthorn has been shown to render
impressive radioprotective effects [15]. It has been exploited
extensively in Indian and Tibetan system of medicine for treat-
ment of several ailments like circulatory disorders, ischemic
heart disease, hepatic injury and neoplasia [16–17]. It is
known that the quantum of free radicals generated by heavy
flux of low LET radiation like gamma rays (10 Gy) is very
high. The antioxidant defence system operating in the cellu-
lar milieu or the presence of exogenous radioprotective anti-
oxidants can not counter these free radicals in a major way.
Therefore it was considered necessary to investigate some
other mechanisms like compaction and modulation of chro-
matin organization as additional mechanism that may help
to account for the degree of radioprotection observed during
in vivo studies. Such insight may help the development of
more effective and less toxic radiomodifying agent.
Materials and methods
Chemicals
Chemicals were procured from different sources: tertiary
butyl hydroperoxide (tB-OOH), triton-X-100, calf thymus
DNA, spermidine hydrochloride, penicillin, streptomycin,
agarose from Sigma Chemical Co., St. Louis, MO, USA;
Propidium iodide from Molecular probes, USA; RPMI-1640
from Hyclone, Germany; fetal calf serum from Biological In-
dustries, Israel. Other chemicals of standard make and pu-
rity were used.
Plant material and preparation
Institute of Himalayan Bioresource Technology, Palampur,
Himachal Pradesh, India collected fresh berries of H. rham-
noides from Himalayan ranges (altitude 3000–4000 m).
Known quantity of the washed and shade dried berries were
extracted using absolute alcohol and triple distilled water
(50:50, v/v; three changes) and the final extract was lyophi-
lized, weighed and stored at 4°C. RH-3 (code name of this
product) was studied for its radioprotective properties in our
Laboratory. RH-3 was diluted in tripled distilled water for ob-
taining desired concentrations and the dose expressed in µg
refers to the weight of dried RH-3.
UV-Vis spectroscopy and HPLC profile of RH-3
The absorbance of RH-3 in UV and visible range was moni-
tored in 100% methanol using Chemito spectrophotometer.
HPLC profile of RH-3 preparation was obtained using C-18
Bonda pack silica column (Waters HPLC system) and mo-
bile phase having Methanol: water (70:30 v/v).
Irradiation
Both in vitro and ex vivo samples as per the requirement were
delivered different doses of gamma radiation by 60Co gamma
chamber procured from BRIT, India. The dose rate during the
course of experiments was about 1.786 Gy/sec.
Isolation of thymocytes and their culture
Randomly selected 6–8 week old Strain ‘A’ male mice were
sacrificed by cervical dislocation, dissected and abdominal
cavity was perfused with 0.9% NaCl; thymus was removed
and visible blood clots were segregated carefully. The thymic
lobes were finely minced and gently crushed using the plunger
of a syringe and the resulting cell suspension was passed
through a 25-gauge needle to avoid cell aggregates. For
studying the nature of binding between RH-3 and chroma-
tin, the thymocytes were suspended in RPMI-1640 contain-
ing streptomycin, penicillin and with 10% FCS and kept in a
CO2 incubator at 37°C.
Treatment to thymocytes
Thymocytes (2 × 106 cells/ml) were suspended in standard
buffer saline and centrifuged at 200 × g for 10 min at 4°C.
Supernatant was discarded and the pellet was suspended in
1 ml of tB-OOH (200 µM) or standard buffer saline (SBS).
The suspension in SBS was exposed to gamma radiation
while maintaining at ice temperature. In either case the sus-
pension was incubated at 4°C for 30 min and the cells were
pelleted by centrifugation (200 × g/10 min, 4°C). The result-
ing supernatant was decanted and the cells were treated with
SBS to wash off traces of tB-OOH if any. The cell prepara-
tion was used for assaying the DNA damage either by single
cell gel electrophoresis or alkaline halo assay. Antioxidants

(butylated hydroxy toluene, 200 µM), Spermidine hydrochlo-
ride (varied concentrations) and RH-3 (different concentration)
as per the requirement were added 5 min prior to tB-OOH treat-
ment or irradiation. For post-irradiation studies the extract
was added to thymocytes 10 min after irradiation.
Isolation of thymocyte nuclei
Nuclei from thymocytes were prepared following Warters
and Lyon [19]. Thymocytes (2 × 106 cells/ml) were suspended
in permeabilization buffer (1 mM KH2PO4, pH 8.0, 150 mM
NaCl, 5 mM EDTA) and immediately pelleted and resus-
pended in permeabilization buffer along with 0.3% Triton-
X-100 for 20 min at 4°C, till 100% of the cells turned trypan
blue positive. Thereafter cells were pelleted and resuspended
in high salt buffer (1 mM KH2PO4, pH 8.0, 150 mM NaCl,
5 mM EDTA) for 10 min at 4°C and pelleted. The pelleting
and resuspension in the same buffer was repeated to get nu-
clei free from cytoplasm as was confirmed microscopically
[20] and were given various treatments as per the experimen-
tal requirement.
Effect of relaxation buffer
The dependency of salt concentration on RH-3 mediated
chromatin condensation was studied in thymocytes. After
treatment with RH-3 (150 µg/ml) thymocytes were incubated
in relaxation buffer (1 mM KH2PO 4, pH 8.0, 5 mM NaCl,
5 mM EDTA) for 20 min at 4°C and thereafter processed for
assaying the DNA damage.
Reversibility/irreversibility of the condensation
The thymocyte after different treatments were resuspended
in complete medium and kept in CO2 incubator at 37°C for
different time periods as per the experimental requirement.
Subsequently the cells were pelleted and resuspended in stand-
ard buffer saline and exposed to radiation dose of 20 Gy and
subjected to single cell gel electrophoresis.
Comet assay
DNA strand breaks in individual cells were detected using
single cell gel electrophoresis [21]. Thymocytes were sus-
pended (2 × 104/ml) in 0.5% solution of low melting agarose
in standard buffer saline and immediately pipetted on to slides
pre-coated with normal agarose (0.1%) and spread uniformly.
The slides were put on steel plate cooled from below by ice
cubes, to accelerate gelling. After 5 min the slides were im-
3
mersed in ice cold lysis buffer (2.5 M NaCl, 100 mM EDTA,
10 mM tris, 1% sodium lauryl sarcosine, 5% DMSO and 1%
triton-X-100, pH 10) and left in refrigerator for 30 min. The
slides were transferred to an electrophoretic tray containing
freshly prepared alkali buffer (300 mM NaOH and 1 mM
EDTA, pH 13) and maintained for 20 min and the slides were
exposed to a constant voltage of 1.5 V/cm for another 20 min
under similar conditions. The slides were removed from elec-
trophoretic tray and washed with 0.4 M tris pH 7.5 three times
and DNA was stained by treating with 200 µl of propidium
iodide (50 µg/ml) in SBS (pH 7.4) for 20 min. DNA was
visualized under LEITZ fluorescence microscope and mi-
crophotography was done using CCD camera. The length of
the comet tail was measured using micrometer and at least
100 comets were measured for each treatment and averaged.
Alkaline halo assay
Thymocytes embedded in low melting point agarose on mi-
croscopic slides were incubated in alkali buffer (0.3 M NaOH
and 1 mM EDTA; pH 13) for 20 min at 4°C and were directly
washed in neutralization buffer (0.4 M tris, pH 7.4), without
being subjected to electrophoretic conditions. This avoided
wide diffusion of the unwound DNA [22]. The propidium
iodide bound intensely stained and intact chromatin mass
formed the central core while the broken DNA fragments
constituted the diffusely stained peripheral halo. Measure-
ments were done from center of the core to any point on the
perimeter of the halo.
Thermal denaturation studies
Different concentrations of the RH-3 were added to a fixed
concentration of calf thymus DNA (1 µg) and mixed gently
and thereafter heated at a rate of 2°C /min using GBC 916
spectrophotometer having ‘DNA melt software’. Buffer blank
and RH-3 blanks were also heated to observe any tempera-
ture dependent variation in absorbance. Change in Tm (when
50% of the DNA melted) was recorded.
Cell viability
Cell viability in the thymocyte preparation was determined
by mixing 10 µl of cell suspension with an equal amount of
0.4% trypan blue in Hank’s balanced salt solution and count-
ing the cells on a hemocytometer under light microscope.
Cells that did not exclude trypan blue were considered as non-
viable. Five hundred cells /treatment were counted and per-
centage viability was calculated.
4

Spectrophotometric measurements A

Abs
3.000
3.000
Abs
Absorbance was measured using Chemito 2500 spectropho-
tometer. All estimations were expressed, as percent inhibi-
tion with respect to control, which was considered as zero
inhibition.

Scavenging of hydroxyl radicals

The quenching of radiation (20 Gy) generated hydroxyl radi-

cals by RH-3 was studied in vitro using 2-deoxyribose degra-
dation as marker assay [18]. Briefly, 2-deoxyribose degrades
on interaction with hydroxyl radicals generated by irradia-

0.000

0.000
tion and forms thiobarbituric acid reactive substances which

200.0

250.0

300.0

350.0

400.0

450.0

500.0

550.0
in the presence of thiobarbituric acid under acidic conditions
give a pink colored chromogen monitored at 535 nm spec- Wave Length (nm)
trophotometrically.
B
Chelation of iron ions

The metal chelating ability of the RH-3 was assayed by quan-

tifying the concentration of free Fe2+ using chelating agent 2,2′
bipiridyl [13]. Briefly 2-2 bipiridyl, a specific indicator of
ferrous ions produces a pink colored chromogen, which was
monitored at 525 nm spectrophotometrically.

Results
Fig. 1. (A) Absorbance profile of RH-3 (dissolved in 100% methanol)
UV-Vis spectroscopy and HPLC profile monitored both at UV and visible range. (B) HPLC profile of RH-3 (dis-
solved in methanol) using mobile phase (methanol:water, 70:30, v/v).

Spectrophotometric profile of RH-3 exhibited prominent

peaks at 210 and 274 nm and a broad peak at 355 nm (Fig.
1A). HPLC profile under conditions described in material and
methods section showed the presence of at least 13 different
constituents (Fig. 1B).
DNA damage and comet assay
to that of the control thymocytes (Figs 3D and 3E). Standard
free radical scavenger, butylated hydroxy toluene (25 µM),
inhibited the formation of radiation (20 Gy) induced comet
tail (Fig. 3F). Spermidine, a known chromatin compacting
agent, at any of the concentrations tried, (2, 5 and 10 mM)
did not prevent the formation of comet tail (Fig. 3G).

Radiation (20 Gy) or tB-OOH (200 µM) alone induced ap-
preciable amount of strand breaks (Figs 2, 3A, 3B and 3C)
in the thymocyte DNA as was evident by the length of the
comet tail (27 ± 2.4 and 32 ± 2.5 µm respectively) as com-
pared to control thymocytes (6.7 ± 1.2 µm). Treatment of
thymocytes with increasing concentrations of the RH-3 re-
sulted in gradual reduction in the tail length. The strand breaks
induced by radiation or tert butyl hydroperoxide were most
effectively prevented by 100 or 120 µg/ml of RH-3 respec-
tively as indicated by the comet tail which was almost similar
Chromatin condensation
RH-3, beyond a concentration of 150 µg/ml, compacted the
chromatin appreciably as evaluated by the lack of comet tail
(strand breaks) and appearance of intensely stained circular
bodies (Fig. 3H) which were resistant to radiation dose of
1000 Gy. The study to reveal the effect of time on interac-
tion between RH-3 and chromatin revealed that a concen-
tration of 150 µg/ml or more of RH-3 achieved complete
interaction with the chromatin and was very fast. Interaction
 5

Fig. 2. Effect of different concentrations of RH-3 on radiation or tertiary

Fig. 3. Thymocytes treated with different compounds and subjected to sin-
butyl hydroperoxide induced strand breaks studied using single cell gel
gle cell gel electrophoresis and were viewed at 400× under a fluorescent
electrophoresis. The length of comet tail, a measure of DNA damage, was
microscope. (A) Control cells without any treatment, (B) 200 µM tert butyl
measured from 100 individual cells from each treatment group under mi-
hydroperoxide, (C) 20 Gy of gamma radiation, (D) 100 µg/ml of RH-3 +
croscope (400×), averaged and expressed as tail length ± S.D. All experi-
20 Gy, (E) 120 µg/ml of RH-3 + 200 mM tert butyl hydroperoxide (F) 25
ments were repeated 3 times.
µM of butylated hydroxy toluene + 20 Gy, (G) 10 mM spermidine + 20 Gy
gamma radiation, (H) 150 µg/ml of RH-3 + 20 Gy.

between RH-3 and chromatin for 5 min rendered the latter Effect of the extract on thermal denaturation of DNA
highly compacted (Fig. 3H). Post-irradiation treatment with
RH-3 (150 µg/ml or more) also revealed a condensation pat- Slow heating of the calf thymus DNA dissolved in standard
tern similar to that of pre- irradiation treatment with RH-3. buffer saline containing 14 mM NaCl increased the absorb-
The condensed chromatin in post-irradiation treatment group ance of UV light and the Tm was found to be 74 ± 2°C. As
was resistant even to very high doses of gamma radiation as the concentration of RH-3 increased (1:0.5–1:1) the Tm was
was observed in case of pre-treatment group (20–1000 Gy). lowered and shifted towards left by 2 and 5°C respectively.
The RH-3-induced condensation of chromatin in nuclear However concentrations above these ratios (1:8 and 1:16)
preparation was similar to that of the whole cell preparation. induced strong shift towards right by 10 and 21°C corre-
Incubation in relaxation buffer did not reverse the RH-3-in- spondingly (Table 1).
duced condensation. The RH-3- chromatin interaction studies
have revealed that the interaction of RH-3 at lower concen-
trations (< 100 µg/ml) was reversible, since the formation of
comet tail could be observed due to the exposure to a radia-
tion dose of 20 Gy, 24 h after the post RH-3 incubation.
RH-3 at higher concentrations (150 µg/ml or more) ren-
dered irreversible compaction as radiation dose of 20 Gy
could not induce tail formation even after 24 h after the drug
treatment (Fig. 4).

Halo assay

The alkaline halo assay also revealed the compaction of chro-

matin by treatment with the RH-3 (Figs 5A, 5B and 5D). As
observed during comet assay, spermidine pre-treatment did not
reduce the girth of halo. However the pre-lysed thymocytes
embedded in agarose gel on treatment with 20 mM of sper-
midine revealed a condensation pattern similar to apoptotic
bodies (Fig. 5C).
Fig. 4. Effect of varied concentrations of RH-3 on reversibility of the con-
densation. Thymocytes exposed to varied concentrations of RH-3 were
incubated in complete medium (RPMI-1640) for 24 h and subsequently
exposed to 20 Gy. The comet tail length was measured from at least 100
cells and averaged and expressed as tail length ± S.D.
6

Table 2. Effect of varied concentrations of RH-3 on viability of thymocytes

6h 12 h 24 h

Normal control 98% 96% 94%

20 µg/ml 98% 97% 98%
40 µg/ml 96% 94% 93%
80 µg/ml 94% 93% 93%
150 µg/ml 95% 93% 92%
200 µg/ml 94% 95% 94%

Thymocytes with varied RH-3 treatments were incubated in complete me-

dium (RPMI-1640) for different periods. Subsequently at different time
points 10 µl of cell suspension was mixed with equal amount of trypan blue
in Hank’s balanced salt solution and observed under light microscope for dye
exclusion. At least 500 cells were counted and expressed as % viability.

Fig. 5. Thymocytes treated with 150 µg/ml of RH-3 were exposed to 20

Chelation of iron ions
Gy gamma radiation and then subjected to alkaline halo assay. (A) Control
cells, (B) irradiated (20 Gy), (C) spermidine (25 mM) + 20 Gy, (D) RH-3 RH-3 failed to elicit any iron chelating potential at any of the
(150 µg/ml) + 20 Gy. concentrations tried here (from 20–1000 µg/ml) (Table 3).

Cell viability
Treatment with RH-3 did not affect the cell viability (Table
2). No concentration of the RH-3 tried here elicited any sig-
nificant loss in cell viability (> 93%; p < 0.05) observed at
different time intervals.
Scavenging of radiation induced OH radicals
RH-3 inhibited degradation of 2-deoxy ribose in a dose de-
pendent manner and IC50 value was 500 µg/ml, whereas for
reduced glutathione the IC50 value was 0.56 mM (Fig. 6).
Lower concentrations of RH-3 elicited only mild scaveng-
ing ability which increased with increasing concentrations
of RH-3. Beyond 100 µg/ml RH-3 scavenged OH radicals
significantly and at a concentration of 1000 µg/ml it was
67%.
Discussion
In irradiated cells the redox reactive Fe2+ are known to in-
crease and participate in Fenton reaction generating hydroxyl
radicals [23]. Any agent which can intercept free radicals will
be significantly effective in minimizing radiation induced
damage [24]. The inhibition of 2-deoxy ribose degradation
is a simple and reliable technique to assess the ability of an
agent to scavenge the radiation-mediated production of hy-
droxyl radicals [18]. RH-3, a preparation of H. rhamnoides
already has been reported to render protection in vivo against
radiation-induced mortality [15]. This study under in vitro
conditions revealed that RH-3 is having potential to scavenge
hydroxyl radical at higher concentrations (> 500 µg/ml) and
yielded 67% inhibition of 2-deoxyribose degradation at a
concentration of 1000 µg/ml. Possibly higher concentrations
could achieve further inhibition but were not attempted for

Table 1. Effect of varied concentrations of RH-3 on melting temperature Table 3. Effects of varied concentrations of RH-3 on chelation of Fe2+
(Tm) of calf thymus DNA
Concentration of the RH-3 % ( 2-2′ bipiridyl-iron) chromogen
DNA:RH-3 ratios Melting temperatures (Tm)
0 µg/ml 100 ± 2
1:0 74 ± 1.5°C 20 µg/ml 104 ± 2.5
1:0.5 72 ± 0.5°C 50 µg/ml 104 ± 3.2
1:1 69 ± 1.5°C 100 µg/ml 109 ± 3.6
1:8 84 ± 2.0°C 200 µg/ml 111 ± 2.5
1:16 91 ± 1.5°C 500 µg/ml 107 ± 2.5
1000 µg/ml 106 ± 4.6
Different proportions of RH-3: calf thymus DNA were taken in 14 mM SBS
in a quartz cuvette and heated gradually and change of absorbance was Inhibition of Fe2+-bipiridyl complex (chromogen) was calculated as percent
monitored spectrophotometrically. Tm was calculated from linear steep part of control, which was considered as zero inhibition. Results were expressed
of the absorbance profile and its mid point was considered as Tm. as mean ± S.D. of 4 parallel experiments.

the lack of practical utility. Lower concentration (< 150 µg/
ml) elicited only 25% inhibition (Fig. 6). The optimal dose
of RH-3 (30 mg/kg.b.w.) for radioprotection in vivo [15] may
therefore not be able to account for OH radical scavenging
in a major way. Therefore radiation protection achieved in
vivo may not be explainable by this mode of action alone.
In the present study 2,2′-bipiridyl assay has revealed that
RH-3 did not chelate ferrous ions (Table 1). Since tert butyl
hydroperoxide induced DNA strand breaks have been asso-
ciated with the generation of hydroxyl radicals at the specific
sites it can be blocked only by Fe2+ chelation or OH radical
scavenging provided the agent was located physically close
to DNA [25]. The ability of RH-3 to inhibit the tert butyl hy-
droperoxide induced strand breaks (Figs 2 and 3) and the in-
ability to act as Fe2+ chelator suggest that RH-3 rendered
protection by modulating the chromatin organization and
scavenging the hydroxyl radicals and not by chelating Fe2+.
In radiation-induced damage and apoptosis, DNA strand
breaks contribute significantly. The damage could be appre-
ciably mitigated if free radicals besides being scavenged are
not permitted physically to interact with the target molecules
like DNA. In fact a number of synthetic and natural molecules
have been reported to protect the cellular DNA from radia-
tion induced damage by reducing access to free radicals [26,
27]. To investigate this aspect comet assay has gained a lot
of importance owing to sensitivity and simplicity of the tech-
nique [21]. RH-3 during ex vivo experiments, demonstrated
prevention of radiation induced strand breaks in a dose de-
pendent manner and maximum effect was achieved at a con-
centration of 100 µg/ml (Figs 2 and 3). Higher concentrations
Fig. 6. Effect of different concentrations of RH-3 on scavenging of radia-
tion induced OH radicals. % inhibition of chromogen was calculated with
respect to control, which was considered as zero inhibition. Results were
mean ± S.D. of 4 parallel measurements. P value was < 0.05 when com-
pared with control. Standard hydroxyl radical scavenger, reduced glutath-
ione, rendered 50 % inhibition at a concentration of 0.56 mM.
7
of RH-3, even on exposure to very high radiation doses did
not allow the appearance of the strand breaks as was adjudged
by the absence of tail and presence of intact core chromatin
(Fig. 3H). In fact inherent DNA bound proteins (histones) and
agents like polyamine have been shown to protect the DNA
from radiation induced strand breaks by modulating the chro-
matin organization [27]. Under in vivo conditions the con-
centration of the active principle of RH-3 that may be able
to reach the critical cellular sites will indeed be quite low and
may neither completely scavenge radiation induced free radi-
cals nor protect DNA completely by compacting the chroma-
tin. Therefore at low concentration of RH-3, as would be
applicable to ex vivo experiments done here, the antioxidant
properties in combination with chromatin compaction might
explain for the protection of DNA and the cells. At higher
concentrations the mechanism of compaction of DNA as seen
under ex vivo conditions may contribute towards radiopro-
tection much more than free radical scavenging.
The post-irradiation treatment with high concentrations of
RH-3 (150 µg/ml or more) resulted in dense compaction of
chromatin. It is possible that molecules present in RH-3 may
induce DNA inter-strand links and DNA-protein cross links
to achieve the condensation. In fact even few DNA-protein
cross-links are known to inhibit appearance of strand breaks
induced by radiation or chemotherapeutic agents [28]. The
short duration (less than 5 min) provided to RH-3 molecules
to interact with irradiated chromatin in the cells (ex vivo)
minimized the possibility of DNA repair because the repair
enzymes may not function with compacted DNA. The revers-
ibility/irreversibility of interaction also depended on persist-
ence of the links between RH-3 molecules with chromatin.
At higher concentrations the interaction remained irrevers-
ible while at lower concentrations it was reversible. The con-
centration dependent reversibility/irreversibility in fact needs
to be investigated. The reversible condensation might be play-
ing a role in radioprotection where as irreversible condensa-
tion would lead to cell death and could be exploited for cancer
management.
The omission of electrophoretic procedure (current flow)
during comet assay, permitted increased sensitivity of dam-
age detection since under ‘no electric field’ conditions, the
small fragments migrate radially and will be present around
centrally located condensed chromatin. This prevents too
broad diffusion of broken DNA strands [22]. The results of
halo assay corroborate the findings revealed by comet assay
regarding RH-3 induced chromatin condensation (Fig. 5).
Spermidine, a known compacting agent of DNA in vitro did
not elicit condensation of cellular chromatin and protection
against radiation induced strand breaks at any of the concen-
tration used (2, 5 and 10 mM) (Fig. 3G). These results are in
corroboration with the earlier reports regarding the inability
of polyamines to protect cellular chromatin [29]. However
when the agarose embedded thymocytes were first lysed and
8
then exposed to 20 mM of Spermidine, a unique condensa-
tion pattern similar to that of apoptotic bodies was observed
(Fig. 5C). These results support earlier findings that Sper-
midine can enter the cell in very limited manner [29]. The
mechanism of the formation of round apoptotic body like
structures need to be revealed by further studies.
Thermal denaturation assay is an efficient method to study
the interaction of any agent with the DNA and was exploited
here to investigate the ability of RH-3 to interact with the
DNA. Lower concentrations of RH-3 shifted the Tm towards
left, probably due to relaxation of supercoiled DNA. Higher
concentrations shifted Tm towards right, probably due to con-
densation of fully relaxed DNA into a positive supercoil form
(Table 2). The relaxation of the chromatin at lower concen-
trations and condensation at higher concentrations is a char-
acteristic feature of intercalators [30]. Therefore it appears
that mode of action of RH-3 to achieve chromatin conden-
sation includes intercalation. At higher concentrations of RH-
3 chromatin condensation is instantaneous being achieved in
less than 5 min of incubation (Fig. 6). This can permit the use
of such compound for protection against planned radiation
exposures. The consequences of higher concentration in-
duced chromatin condensation need to be understood with
special reference to DNA synthesis, transcription and normal
physiological functions (cell cycle progression and apoptosis
etc.). The HPLC profile of RH-3 revealed presence of at least
13 compounds. It is therefore necessary to isolate and charac-
terize the active fractions responsible for chromatin conden-
sation, free radical scavenging and other associated activities.
Acknowledgements
The authors gratefully acknowledge the support provided by
Dr. T. Lazar Mathew, Director, INMAS during the course of
this study. The authors are also thankful to Dr. A.K. Sinha,
Institute of Himalayan Bioresource Technology, Palampur,
H.P. India for providing the crude plant extract.
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