Indian Journal of Experimenta l Biology Vol. 40, May 2002, pp. 525-530

Protective effect of RH-3 with special reference to radiation induced micronuclei in mou se bone marrow

P K Agrawala & H C Gae l'"

Radiat ion Biology Di vision, Institute of uclear Medicine and All ied Sciences, Lucknow Marg, Delhi 110054, IndiJ

Reeeil'eti I ] Oe/Ober 2000; revised] j Olll/or)' 2002

Elfect uf pre-irradiation admini stra tion of different doses of RI-I-3, the herbal preparation of an Indian Ill edi c in ~tl plalll Hippoplwe ,." aJIIl/oides, 30 min before lOGy whol e body gamma irrad iation was studi ed. Doses between 25 to 35 mg/kg body wt. were found to render > 80 % survival in mice. In order to in ves ti gate whether RI-I -3 protec ted against radiation in­duccd gcnotox ieit y, mice werc admini stcrcd diffcrent doscs of RII-3, 30 min beforc 2 Gy dose and compared with un trca ted, RI-I-3 treated and irradiatcd controls. Thc bone marrow cell s wc rc co ll ec ted at different timc interva ls fo llow ing vari ous trca tmcnts and processed fO I· scoring micronuclei (MN). Admini strat ion of RI-I -3 alonc did not cnhance the MN frcqucncy as compared to the contro l, and racliJti on dose of 2 Gy signifi cantl y cnhanced the MN frequcncy (3.1 %, P < 0.0 I ). Pre­irradiation treatmcnt with RI-I -3, however, reduccd thc rad iat ion induccd M N frequency in a drug dose dcpendent man ncr suggcsting its rad ioprotect ive cffi cacy. The prot cc ti vc effect of RI-I-3 0 11 radiati ol1 induccd perturba ti ons in cell cyc lc pro­gress ion 11 :1 , st udicd flowcytoillctr ica ll y in mousc bone marrow cc ll s. RI-I -3 trcatmclll (30 mg/kg body wt.) enhanced DNA synthcsis (S-phasc) in unirradiated controls Jnd also countcrcd rad iat ion induccd depression of S-phase to facil ita lc rcplcn­ishmcnt of cells lost due 10 radial ion injury.

A large number of molecular drugs have been sc reened for their radioprotec ti ve efficacy, however, because of the inherent toxicity at useful concentra­tion, none of them could f ind clinical acceptance I.

Plant products are known to have many compounds of medicinal value. Therefore th ere is an emerg ing in ter­est during last several years in the plant products for their radioprotecti ve efficacy~-5

Hippop/zae rhallllloides (Elaegnaceae), common ly known as Sea buckth orn can withstand extreme

conditions (-43° to +40°C, pH 5.8 to 8.3 and high salt concentrat ions) and has been ex tensively used in Indian medicine system 'Ayu r veda' and also in the preparation of modern pharmaceuti ca ls, cosmetics, animal feed, sport and health drinks, etc. H. rhalll­lIoides has been reported6 to contain a large number of ant ioxidants l ike vitami ns A, C and E, hydro lyzable

. 7 f'l 'd 8 I 6 I· k I ' tanntns, avonol S , trace e ements t ' e se enlum, zinc, su lfur an copper. It has been reported to inhibit Ca2

+ infiux9 and transfer of electron from cytochrome­C to oxygen in the respiratory chain 7 and to protect against lipid perox idation lO

, atherosclerosis 10, eye burns II and improve gastric function. Compounds

*Correspondcn l aUlhor: Tcl: 9 1-11- 397008 1; Fax: 91- 11 -39 19509 E-mail: racl biol @nda.vsl1l.ncl.il1 heg @driI1Ill Cl. rcl1 .nic.i l1

having antioxidant properties are known to provide protection against radiati on and other ox idat ive stresses. These compounds may dec rease locali zed oxygen concentration, prevent f irst chain initiation by scavenging initial radical s and/ or bind metal ions in forms tha t will not generate reactive oxygen species further. Vitamins A, C and E have been shown to in­hibitlipid peroxidation l2

, scavenge free radicals 13, anci

protect aga inst radiati on induced chromosomal aber-. d' I ' d . 14 ts S I . ration s an I11lcronuc eus III uctlon " . e en lum pre-

sent in H. r/wlIIlloides may help in biosyn thes is of glutathione peroxidase l6

, which is crucia l for the de­composition of lipid hydroperoxides.

Flavonoids have been shown to reduce the fre­quency of radiation-i nduced micronucle i in mouse bone malTow3

. Hydrolyzable tannins are potent inh ibitors of hydroperoxide production and tumor promotion l 3

. Calcium ions play a vital role in ox idative stress induced cell dea th l7 and in radiati on injury' ~ . Owing to these propert ies RH-3, a preparat ion of H. rhall1l1oides was considered suitable for studying its radioprotective action .

The rodent bone marrow micronucleus (MN) assay has been widely used for the detection of the mutagenic, carc inogenic and genotox ic potenti al of various chemica l and physical agents including ioni z­ing rad iations I 9

-2 1

. For genotox icity stud ies, the MN

526 INDIAN J EXP BIOL. M A Y 2002

assay was preferred over the dicentric analysis be­cause it is simple, reli abl e, and rapid22

. MN assay us­ing conventional Giemsa staining enab les the di s­cri minati on between mature and immature erythro­cytes (normochromatic erythrocytes, NCEs; poly­chromatic erythrocytes, PCEs respecti ve ly). The PCE/NCE rati o remains close to 1 in hea lth y anima ls and provides va lu able information on the bo ne mar­row cy totoxic properti es of the test agents.

Ionizing radi ati on causes transient de lay in cell di­vision th at may include G I arres t, S-phase delay, and G2 arrest. The G2/M checkpoint is considered as the most sensitive stage of cell cycle. Radi ation induced G2/M delay is considered an adaptive response of the cell fo r the repair of DNA damage before the cell en­ters to M phase. Agents that eliminate or reduce the G2/M checkpo in t cause reduced DNA repair and therefore enhance rad iosensi ti vity of the cell s23. Since alterations in cell cycle progress ion can alter the rad io sensi tivi ty of cells, agents th at can interfere with the cell cycle regulating machinery can be useful in pro­viding radio sens it iza ti on or protection. Percentage of cells in diffe rent phase of cell cyc le and their progres­sion through different phases can be quantitative ly monitored by fl owcy tometric methods. Haemopoiet ic cells are hi ghly sensiti ve to ioni zing radiati on owing to their high prol ifera ti on rate. Cell cycle checkpoints have been considered important molecul ar targets for modulati on of rad iati on res ponse. Therefore, it was felt necessary to in vest igate the effect of RH-3 on ra­diation induced ce ll cycle perturbat ions in mouse bone marrow cell s.

Material and Methods Anill1als- Inbred strain 'A' male mice (6-8 week

old), we ighing about 25± 3 g were housed in polypro­pylene cages under contro lled conditions (25° ± 2°C and 12 hr photoperi od) and were provided standard pellct food (Hindusthan Lever Ltd. ) and tap water ad IibituIJI. Experimental procedures were adopted as approved by the animal experimentation ethi cs com­mittee and compiled with the standard labora tory guidel ines of the institute.

RH-3- Berries of H. rhallll10ides were co ll ected by Institute of Himalayan Bioresource Technology (CSIR), Palampur (HP), from an altitude of about 12000 ft from Leh region of I ndi a and identi fied by ethnobotanist and processed in our laboratory fo r th e producti on of a patented herbal prepara tion, RH-3.

Irradiation-The req uired doses (mid-line ti ssue dose) were del ivered from a C060 gamma source

(Gamma Cell , Model 220, Atomic Energy, Canada). Dos imetry was carried out with Bald win Farmer sec­ondary dos imeter and Fricke dosimeter. The dose rate reduced from 1.05 to 0.98 Gy/min during the experi­mental peri od. Fresh air was being supp lied into the irradiation chamber contin·uously through a pl astic tube to main tain normal oxygenation.

Dmg Ireotment - RH-3 was di ssolved in double di stilled water to get the des ired concentrati ons. Sham contro ls received normal saline. All the fluid s were administereJ ip and the fina l inj ec ti on volume was kept about 0.2 ml fo r each an imal.

Survival study agaill sl 10 Gy whole body gClll/ll/a irradiotion

A lethai dose of lOGy was selected for whole body survival studies based on the earlier findin gs on LD 50 (30) of strain 'A' mi ce lK

• Eighteen groups of ani ­mals (n = 10 fo r rad iation alone group, I I for 30 mg/kg RH- 3 + 10 Gy, ! 0 fo r 50 mg/kg RH- 3 + lOGy and 5 in all other groups) were treated as described below. Group r constituted the untreated control and received 0.2 ml normal sa line ip, group II an imals recei ved a lethal dose of lOGy whole body ga mma irrad iation; animals in groups III to X wcre ad minis­tered 10, 15, 20, 25, 30, 35, 50 or 100 mg/kg body wt. of RH-3. An imals in groups XI to XVIll rece ived 10 Gy dose, 30 min follow ing the adm ini stra tion of dif­fere nt doses of RH- 3 as in groups III to X. The ani­mal s were observed dai ly till 30 post-treatment days under controlled laboratory conditi ons. The surviva l freq uency was ex pressed as percentage of the initi al

I · I 301h . d·· d numoer In eac 1 group, at post-Ina ;atlOn ay.

Micronucleus assay Forty-eight animals were divided into 16 groups of

3 each and vari ous treatments were done as desc ri bed below:

Groups I and 2 const ituted normal controls and were sacrifi ced either 24 or 36 hr aft er ip Ireatment with 0.2 ml normal sa line. Si mil arl y, group 3 and 4 an ima ls were 2 Gy whole body gamma irradi ated and sac ri ficed 24 or 36 hr later. Animals in groups 5 to 7 were admini stered 20, 30 or 40 mg/kg body wt. of RH-3 and sacrificed 24 hr post-treatment ti me. Ani ­mals in groups 8 to [0 received sa me doses of RH -3 as in groups 5 to 7 and were sacri ficed at 36 hr pos t­treatment time. Animals in groups II to j 3 and 14 to

16 were also admini stered 20, 30 or 40 mg/kg body wt. of RH -3, 30 min . prior to 2 Gy whole body gamma irrad iati on and sac rificed 24 or 36 hr post­irradi ati on time.

)I

AGRA WALA & GOEL: RADIOPROTECTIVE EFFECT OF RH-3 527

Preparalioll of slides-The femur bone marrow cells were ex tracted at desired time intervals with 0.5 ml of PBS and centri fuged once at 1000 rpm before being suspended in FCS. Smears were spread on cleaned glass slides, fixed in methano l for 30 min and air-dried overnight. Slides were stained with 4% Giemsa solu tion dil uted in Sorensen's phosphate buffer, pH 6.8 for 8 min l 9

. Excess stain was washed and the ai r-dri ed sl ides were mounted with glycerol phosphate. For the anal ys is of the frequency of mi­cronucleated polychromatic erythrocytes (MNPCEs) a minimum of 2000 PCEs were scored from each ani­mal. The MN frequencies were expressed as percent­age values. The rati o of PCEs to NCEs was calculated by scoring all the PCEs and NCEs present under the microscope fie ld (100 x magnifi cation). At least 2000 NCEs and all the PCEs we re considered for each ani­n1:11 in var ious treatment groups. Levene 's test was app lied fo r an alysis of va ri ance; P < 0.5 was consid­ered significant.

Cell cycle allC/ly~is

For cell cycle analys is animals were distribu ted in 10 groups of 3 eJch . Group A animals constituted the normal control and were administered 0.2 ml normal sa line ip; animals in groups B, C and 0 were exposed to a dose of 2 Gy. Three groups of animals (E-G) were treated with 30 mg/kg body wt of RH-3 alone and animals in groups H to J received 2 Gy dose, 30 min after the adm inistration of 30 mg/kg body wt. of RH-3. The animals were sacrificed after 4,24 or 48 hr fo llowing various treatments and the bone marrow cells were coll ected in PBS and centrifuged at 1000 rpm for 10 min. The bone marrow cells were treated with 2 ml chilled RBC lysi s buffer contain ing ammo­nium chloride on ice for 3 - 5 min so as to remove the enucleated cells, washed once with 5 ml PBS and fixed in 70% ethanol overni ght at _20De.

Preparatioll of cells for jlolVcytolll etry-Approx imately one million cells from each sample were washed once with PBS at 1000 rpm. The cell pellet was resuspended in 0.5 ml PBS in 12 x 75 mm round bottom po lystyrene FACS tubes and treated with 100pg/ml RNase A for 30 min followed by 25 pg/ml PI in PBS under dark condition for next 30 mi n25

. All the treatments were carried ou t at 37° e. From each sample, data of 10000 cells were acquired and ana lyzed using a Becton Dickinson FACS flow cytometer eq ui pped with 488 nm argon laser and suit­able fil ters. A standard scatter plot was defined based

on the optical properti es (forward scatter and right angle scatter) of the cell s to eliminate cell debris and ce ll aggregates. Percentage of cells in va rious stages of the cell cycle was anal yzed by MOD-FIT software. Student' s t tes t was app li ed to calc ul ate the level of significance and P < 0.05 was considered significant.

Results Survival studies-Whole body irradiation ( lO Gy)

res ulted in 100% mortali ty withi n 10 to 15 post­irradiation days (Fig. I). RH- 3 alone up to a dose of 35 mg/kg body wt did not induce any mortal ity . Treatment with dose of SO or 100 mg/kg body wt of RH-3 resulted in 40 and 100% mortality respectively. Pre- irradi at ion admini st ration of different doses of RH-3 pro tected against 10 Gy radiati on induced mor­tality (Fig. I), max imal survival (> 80%) was ach ieved by a drug dose of 25 to 35 mg/kg body wt. of the drug; both lower or hi ghe r drug doses resulted in decreased survival.

Microlluclells assay- Rad iation induced MN fre­quency and its modulation by pre-treatment with RH-3 has been depicted in Fig. 2 and Table I. Irradiation alone res ulted in a signi ficantly enhanced MN fre­quency (3. 10%; P < 0.0 I) as compared to the control val ue (0.23 %) at 24 hr post- treatment time. Admini­strat ion of any dose of RH-3 alone did not eli cit any increase in the MN frequency (groups 5-7) at 24 or 36 hr post- treatment time interval s. The doses of 20 to 40 mg/kg body '.vt of RH-3 sig ni ficantly (P < 0.05) re­duced radiation-induced MN frequen cy in a dose de­pendent manner. The doses of 30 and 40 mg/kg body wt of RH -3 were most effective and reduced the M frequencies for these two doses did not vary illter se significantly at any of the time intervals studi ed.

120

100

80

~ ~ 60 .~

:> If)

40

20

0

D RH-J I [] RH-3.1 0ayl

,-~ .•

0 10

" ~ ~ "-!

. 0 .-i

l . . ~ ! .} :; .: ~~ "f. e.:.

x , ~; ., ;'j

;0: I~ .~.; . ~ I .!j. ,

15 20 25 30

Doses of RH-3 (mg/kg body wt)

-'I r-;, .. , ' ( ~

-,'. .~ :.

1-~~ ~'

'" ; : , d ~ .,

35 50 100

Fig. 1-Effec t of pre-irradiation administration of clifferenl doses of RH-3 on survival of lOGy whole body gamma irradiatcd micc.

528 INDI AN J EXP BIOL, MA Y 2002

Though the MN frequenci es at 36 hr post-treatment ti me interva l in different trea tment groups were higher than their corresponding MN frequencies at 24 hr, the trend remained similar.

Table 1 depicts PCE/NCE ratios at 24 and 36 hr post- trea tment time interval s along with the MN fre­quenc ies in different trea tment groups. The PCE/NCE ratio dec lines drast ica ll y in response to many cy to­tox ic stimuli and accordingly 2 Gy irradi ation reduced the PCE to NCE rati o sign ifi cantl y (P < 0.05 ) as com­pared to the control (0.6 1 for 2 Gy compared to 0.96 in case of the control at 24 hr post-treatment time in­terva l). RH-3 alone did not induce any bone marrow cytotox icity and the PCE/NCE values remained close to control values at 24 and 36 hr time interval s. The PCE/NCE rati o at 24 hr in different treatment groups rema ined hi gher than their corresponding PCE/NCE rati o at 36 hr time interva l. Pre-treatment with any dose of RH -3 decreased the radiation induced bone marrow cy totox icity significant ly at both the time in­terva ls studied as observed by the hi gher PCE/NC E rati o in RH -3 pre-treated irradiated groups.

Table I- Effe ct of differen t doses of RH -3 on rad iation-induced MN freq ucncy and bone marrow cytotox icit y

[Values are mean ± SD of 3 animals in each gro upl

Group

Cont rol

RH-3 (mg/kg) alone

20

30

40

Radiat ion alone

2 Gy

RH-3 (mg/kg)+ 2 Gy

20 + 2 Gy

30 + 2 Gy

40 + 2 Gy

Time intcrva l

(hr)

24 36

24 36 24 36

24 36

24 36

24 36

24 36 24 36

No. of MNPCEs observcd

(9'< )

0.23±0.07 O.22±O.OS

0.22±0.06 0.23±0.07 0.20±0.OS 0.2 1±0.OS 0.2S±0.06 O.27±O.OS

3. 10±0.90 3.20±0.9S

I.S6±0. 13 1.70±0.16 1.2S±0. 13 I AO±O. I I IAS±O. I S 1.60±0. IS

PCEs/NCEs

0.96±0.OS 0.96±0.07

0.97±0.06 0.96±0.OS 0.9S±0.06 0.9S±0.07 0.9S±0.04 0.96±0.OS

0.6 1±0.03 0.S6±0.04

0.S6±0.06 0.S7±0. 10 0.89±0.09 0.S9±0.OS 0.S4±0.OS 0.SS±0.07

Cell cycle onolysis-2 Gy irradiation red uced markedly the S-phase cell population that corrobo­rates earli er findings2S

. 26 (Fi g. 3). The S-phase cell population decreased to minimum at 24 hr post­irradiation time. Appreciable recovery was, however, witnessed at 48 hr post-irrad iation time peri od. Pre­irradi ati on adm ini stration of RH-3 protected against the rad iation induced suppress ion in S-phase and the percentage of cell s in S-phase were co mparable to that of control at 48 hr post-irrad iation time period .

Irradia ti on resu lted in a transient delay in progres­sion of cell cyc le through G2 block, wh ich persisted up to 48 hr post-irradiation time (Fi g. 4). The effect of RH-3 alone on G2 chec kpoint also witnessed a similar trend . Pre-irrad iati on adm ini strat ion of RH-3 did not alter the radi at ion induced G2 delay.

DisclIssion Radi oprotection indeed, is a wholeso me ac ti on ,

which may include scavenging of free radical s, pro­tecti on of cellular and sub-cellul ar entiti es especiall y aga inst ox idati ve damage, repa ir of the target mole­cules like DN A, proteins etc, and restorati on of ce ll proliferati on. Compounds having such properti es can offer protection aga inst radi ati on damage.

Admini strat ion of 25 to 35 mg/kg body wt of RH -3, 30 min before whole body lethal irradiation ( 10 Gy) provided max imum survival (Fig. 1). Poss i­bl y, the lower doses were not able to generate the ra­dioprotective action to the full ex tent and hence were less effec ti ve. The radioprotective mechan isms initi­ated by hi gher doses (>35 mg/kg body wt) ,"ere though already functional at their peak ye t could have

3.

1. 0

: con r h-3.20 r h - 3,30 rh-3,40

Treatment

Fig. 2-The dose res ponse curve for mieronuc lell s frequency in bone marrow ce ll s of whole body gamma irrad iated (2 Gy) mice, treated with diffe rent doses of RH -3, 30 min before irradiation .

AGRA WALA & GOEL: RADIOPROTECTIVE EFFECT OF RH -3 529

generated some toxIcIty concurrently by disturbing certain metabolic pathways, thereby negating the ra­dioprotecti ve efficacy. Infact, hi gher concentrations (>50 mg/kg body wt) of RH-3 alone induced about 40% mortality (Fig. I).

The pre ent study dealing with different doses of RH-3 (Fi g. 2 and Table 1) in protecting against radi a­ti on-induced genotoxici ty as observed by MN fre­quency also indicate that 30 to 40 mg/kg body wt. doses were maximally effective. These results cor­roborate the whole body survi va l studi es (Fig, I) where maximum rad ioprotec ti on was ac hi eved with the dose of 25 to 35 mg/kg body wt of RH-3. Treat­ment with RH -3 alone neither enhanced the MN fre­quency nor reduced the peE/NeE ratio compared to control, in dicating the doses used were not cytotoxic.

25

Control ... --

" '8

Time Interval (hr)

Fig. 3-EfTecI of RH-3 pretreatment, (30 mg/kg body wt, -30 mi n) on 2 Gy induced perturbalions in S-phase di stribution or mouse bone marrow cells,

16

12

L. Control .. -

1

2 Gy

i .',. " ,.

Orug+2 .... /"

J .. ','

" ,,-'/

.' / .' , ,,' J/

. " ;";":": :" .:.:":"'~ '- --Dl\Jg

t

O~----L-____ ~ ____ ~ ____ ~ ____ ~ ____ ~ 21. 1.8

Time interval (hr)

Fig. 4-Effcel 0:- RH- 3 pre lreatmenl, (30 mg/kg body wt , -30 min) on 2 Gy induced G2 block in mouse bone marrow ce ll s.

RH-3 pre-treatment al so protected mice against rad ia­tion induced cy totoxic ity and increased the peE/NeE rat io significantly. It is also possible that RH-3 stimu­lates bone marrow cell proliferation and thereby in ­creased the number of peEs. This assumption is sup­ported by the cell cyc le data as discussed later. Since RH-3 contains many anti oxidative compounds includ­ing fla vonoids and vitamins, it may act through a mechani sm in vo lving scavenging of radiation induced free radical s. It is also possible that RH-3 stimulates the cellular repair machinery and substanti all y re­duces the MN frequ ency below th e control va lue, but such mechan ism is yet to be unraveled.

The effect of radiation on cell cyc le progress ion and its modul ation by RH-3 was studied flowcy tomet­ri ca ll y. Rad iation alone induced a transient delay in G2 check point of the ce ll cycle and depleted the S­phase population as reported earli er26

, The S phase cell population in irradiated animals was substantiall y reduced by 24 hr post-irradi ation time (Fig. 3). How­ever, at 48 hr post-irradiation time, the S phase ce ll population was comparable to untreated controls. RH-3 alone or its pre- irradiation treatment increased the number of cells in S phase indicating the proliferative stimulati on by RH-3 treatment. This enhanced prolif­eration cou ld contribute towards radioprotection by helping the replenishment of radiation induced cell loss.

Radiation induced G2/M delay is important for cell surv i val and is an adap ti ve mechanism to overcome radiation induced DNA damage26

, Before the cells en ter M phase, G2/M delay provides time for DNA repair and reduces the chances of fixation of rad iation damage. Attenuation of G2 block has been shown to increase the frequency of chromosome fragments in human lymphocytes25

. During the present study also irradiation increased G2/M cell popul ation in a time dependent manner reaching to maximum level at 48 hr post-irradiation time (Fig. 4). Interestingly , RH-3 alone also increased the cell population at G2/M phase. In RH-3 treated irradi ated group the G2 frac­tion was comparab le to irradiated group. The increase in G2 cell population in both the groups, however, can be accounted due to different reasons, For irradiated group the repair of damaged DNA warranted G2 de­lay, on the contrary RH-3 treatment enh anced S-phase and therefore increased S-phase population on pro­gress ion which resulted in an increase of G2 popula­tion . This was true when RH-3 was given alone or as pre-irradiation treatment.

530 IND IAN J EXP BIOL, MA Y 2002

Radioprotection of cy togenetic damage and stimu­lat ion of cell proliferation to replenish cells los t due to radiation injury can ex plain to a reasonab le degree the whole body survival against lethal irradiation. The results are preliminary and need further conformati on including mo lecul ar mechani sms in volved in RH-3 mediated radioprotecti ve effects.

Acknowledgment The authors thank S. Singh and J. Prasad for tech­

nical support and JS Adhikari for flow cytometry .

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