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1615 Sharma et al.World Journal of Pharmacy and Pharmaceutical Sciences PROTECTVE ROLE OF LIV.52 AGAINST RADIATION AND CADMIUM INDUCED HAEMATOLOGICAL (RBC) CHANGES IN THE SWISS ALBINO MICE Sharma Ramakant1 and Purohit Rajendra2 1Ramadevi Mahila (P.G.) Mahavidyalaya, Harnathpura (Nua) Jhunjhunu. 2Radiation Biology Lab. Dept. of Zoology, Govt.Dungar college, Bikaner, India ABSTRACT The present study was aimed to evaluate protective efficacy of a herbal drugLiv.52againstradiationandcadmiuminducedHaematological changesintheSwissalbinomice.Theanimalstreatedwithgamma radiationand/orcadmiumchloridewithandwithoutLiv.52were sacrificedbycervicaldislocationatposttreatmentintervalsof1,2, 4,7,14 and 28 days. The values of RBC found to increase up to day-14 in non drug treated groups and day-7 in drug treated groups. Thereafter adecreaseinthevaluewasobservedwithoutreachingtothenormal. Whentheanimalsweretreatedwithradiationandcadmiumchloride simultaneously,synergisticeffectswereobserved.Inalldrugtreated groupsrecoverystartedearlierthanthatin non drug treated groups.The Liv.52 treated animals exhibitedlessseveredamage and early recovery as compared to nondrugtreatedgroups.Thus,itappearsthatLiv.52ispotentenoughtocheck Haematological change in the Swiss albino mice. KEYWORDS: Radiation, Cadmium, Liv52, Haematological change, RBC, Mice. INTRODUCTION Red blood corpuscles (RBC) Inthepresentobservationerythrocytecountdecreasedonexposuretodifferentdosesof gammaradiationandthedepletionwasmorepronouncedwithhigherdosesconfirmsthe previousfindingsofSpalding(1966),whonotedthatthedepopulationofRBCfollowing exposureissomewhatdose-dependent.Thepresentexperimentexhibitedasignificant W WO OR RL LD D J JO OU UR RN NA AL L O OF F P PH HA AR RM MA AC CY Y A AN ND D P PH HA AR RM MA AC CE EU UT TI IC CA AL L S SC CI IE EN NC CE ES S S SJ JI IF F I Im mp pa ac ct t F Fa ac ct to or r 2 2. .7 78 86 6 V Vo ol lu um me e 4 4, , I Is ss su ue e 0 01 1, , 1 16 61 15 5- -1 16 63 30 0. .R Re es se ea ar rc ch h A Ar rt ti ic cl le e I IS SS SN N 2278 4357 *Correspondence for AuthorDr. Ramakant SharmaRamadevi Mahila (P.G.) Mahavidyalaya, Harnathpura (Nua) Jhunjhunu. Article Received on 17 Nov 2014, Revised on 09 Dec 2014, Accepted on 29 Dec 2014 www.wjpps.com Vol 4, Issue 01, 2015.

1616 Sharma et al.World Journal of Pharmacy and Pharmaceutical Sciences depletioninerythrocytecountwhichcontinueduptoday14inthenon-drugtreatedgroups andday-7intheLiv.52treatedgroupsincreasedthereafter,tillthelastautopsyinterval i.e.day28.Norrisetal.(1998)notedthatafterexposuretogamma-radiation,erythrocyte countsindogsreachedaminimumlevelat22days.KohnandFruth(1952)reportedthat decreaseinnumbermightbeduetotheirlossthroughhaemorrhageandradiation-induced injury directly on the cell wall or indirectly via vessel trauma. Sainietal.(1985)alsoobservedsignificantdecreaseinerythrocytecountwhichcouldnot reach the normal level even on 70th day, after the exposure to gamma radiation. Decrease in erythrocytecountsonexposuretoradiationhasalsobeenreportedbyKumaretal.(1982, 1983); Floersheim et al. (1988); Shaheen and Hassan (1991) and Pecaut et al. (2002). Thedecreaseinthenumberofredbloodcorpusclesinthepresentstudymaybedueto defectivehaemopoiesis as well asintravascular red cells damage (Stohlmanet al., 1957). In addition, the shortening oflife span of erythrocytesby radiationmayhave asignificant role inbringing about theerythrocyte depletion. Itis an establishedfact that the depletionin the various blood cell components is largely due to the adverse effects of radiation on the blood-formingorgans.BruesandStroud(1964);Saini(1977)reportedthatwhenbonemarrow becomes totally aplastic and its proliferative capacity and that of other blood forming organs isreducedornullifiedbyheavyirradiationthen"stemcellsinredpulpofthespleenstart dividinganddifferentiatingintoerythrocytesandmyeloblaststocompensatetheperipheral blood cell loss. As it is clear from the kinetics of the red cells, the fall in RBC count after irradiation might be duetochangesinplasmavolume,leakageofcellsthroughcapillariessecondaryto thrombopenia and some instances, by severe haemorrhage(Bond et al, 1965). In the present work,therecoveryofredbloodcellcount,afterirradiationcanbeattributedtothe replenishment from the bone marrow. However, at higher doses the bone marrow also suffers from hypoplasia (Saini, 1977). Cadmium chloride intoxication induces haematological changes in the exposed animals. The presentstudyexhibitedasignificantdeclineintheredbloodcorpusclescontinuouslyupto day-14 in the non-drug treated group II and day 7 in the Liv.52 treated groups. www.wjpps.com Vol 4, Issue 01, 2015.

1617 Sharma et al.World Journal of Pharmacy and Pharmaceutical Sciences DecreaseinerythrocytecountaftertheadministrationofCadmiumhasbeenreportedby many researchers (Kumari and Banerjee, 1986; Prakashet al., 1988c; Mukherjee and Sinha, 1993; Mackova et al., 1996; Yamano et al., 1998). Balaetal.(1994)observedasignificantdeclineintotalRBCcountinthebloodofChanna punctatusexposedtosublethalconcentrationofcadmiumchloride.Decreaseintotal erythrocytecount(TEC)duetosub-lethalexposuretocadmiumwasalsorecordedby Panigrahi and Mishra (1976); Srivastava and Mishra (1979). In their experiment TEC showed unevenincreasingordecreasingtendency.Besidesthis,Weigeletal.(1984)observedan increasednumberofredbloodcellsafter40daysofcadmiumexposure.Assuggestedby Prigge(1978)anincreaseinthenumberofredbloodcellswithoutconcomitanthemo-concentration,whichheobservedafterinhalativeuptakeofcadmiumbyrats,isadirect stimulatory effect of this metal on erythropoiesis. BloomandBloom(1954)andHulse(1959)havesuggestedthreepossibleexplanationsfor the radiation in the number of the different groups of cells following irradiation.(i)There may be a Cessation of mitosis lasting for a variable period.(ii) Thecellmayshownoimmediatevariableabnormalitiesbutdieduringthenextof subsequent mitosis or(iii)Thecellmayundergodegenerationanddiebyamechanismwhichisrelatedtothe process of cell division. In theLiv.52 treated groups declinein RBC waslesser and an early recovery wasalso seen whichmaybeduetoprotectionprovidedbythedrug.Theseresultsareupholdedbythe findings of purohit et al. (2009). MATERIALS AND METHODS EXPERIMENTAL ANIMALS SixtoeightweeksoldmaleSwissalbinomicewereprocuredfromaninbredcolony maintainedinanimalhouseofHAU,Hissar.Theanimalswerekeptinthepolypropylene cages in the departmental animal house of Govt. Dungar College Bikaner .The standard mice feedandwaterwereprovidedadlibitum.Thetemperatureoftheanimalhousewas maintained between 20-25c. www.wjpps.com Vol 4, Issue 01, 2015.

1618 Sharma et al.World Journal of Pharmacy and Pharmaceutical Sciences SOURCE OF IRRADIATION Acobalt-60gammaradiotherapysource(Theratron)ofAECLmakeobtainedfromCanada was used for irradiating the animals in the present investigation. This facility was provided by the Radiotherapy Department of Prince Bijay Singh Memorial Hospital, Bikaner (Rajasthan). Theanimalswereirradiatedat thedoserate of0.97Gy/minute.Thedosewascalculatedat mid point bymultiplying dose rate and tissue air-ratio. The tissue of Swissalbinomice was assumed to be equivalent to human soft tissues. CADMIUM CHLORIDE TREATMENT Cadmiumsaltintheformofcadmiumchloride(SDSChemicals,India)waspreparedby dissolving 20mg ofcadmium chloridein 1000ml of the glass distilled water, thus givinga concentration of 20 and then administered orally in drinking water. Liv.52 Liv.52 drops were procured from Himalaya drug company, Mumbai, India. The drug was fed orallyatthedoserateof0.05ml/animal/daysevendayspriortoirradiationandcadmium chloride treatment till the last autopsy day of experiment. EXPERIMENTAL DESIGN The animals for the experiments were divided into the following groups Group1: (Sham-irradiated animals-normal) Animals of this group were sham-irradiated and served as normal group. Group II: (Cadmium chloride treated animals) The animals of this group were orally fed with cadmium chloride solution at the dose rate of 20 ppm ad libitum in drinking water continuously till the last autopsy day. Group III:(Only irradiated animals) The animals of this group were exposed to sub-lethal doses of gamma radiation from cobalt-60 source. This group was further divided into two sub groups on the basis of radiation dose received: Sub- group III a: 3.0 Gy Sub- group III b: 6.0 Gy www.wjpps.com Vol 4, Issue 01, 2015.

1619 Sharma et al.World Journal of Pharmacy and Pharmaceutical Sciences Group IV: ( Animals treated with radiation and cadmium chloride) Theanimalsofthisgroupwereorallyfedcadmiumchloridesolutionatthedoserate of20 ppm and also exposed to different doses of gamma radiation. This group was further divided into two sub groups on the basis of radiation dose received: Sub- group IV a: 3.0 Gy+CdCl2 Sub- group IV b: 6.0 Gy+CdCl2 Group V:(Animals treated with cadmium chloride and Liv.52) Theanimalsofthisgroupwereorallyfedcadmiumchloridesolutionatthedoserate of20 ppm and were also administered Liv.52 orally for seven days at a dose of 0.05ml/animal/day prior to cadmium chloride treatment and continued up to the last autopsy interval. Group VI:(Animals treated with radiation and Liv.52) The animals of this group were exposed to sub lethal dose of gamma radiation from cobalt-60 source.TheLiv.52wasgivensevendayspriortoirradiationandcontinueduptothelast autopsy interval. This group was further divided into two sub groups on the basis of radiation dose received: Sub- group VI a: 3.0 Gy+Liv.52 Sub- group VI b: 6.0 Gy+ Liv.52 Group VII: (Animals treated with radiation, cadmium chloride and Liv.52) Theanimalsofthisgroupwereorallyfedcadmiumchlorideatthedoseof20ppmand received Liv.52 orally for seven days at a dose of 0.05 ml/animal/day prior to irradiation and cadmium chloride till the last autopsy day of experiment. This group was further divided into two sub groups on the basis of radiation dose received: Sub -group VII a: 3.0 Gy + CdCl2 + Liv.52 Sub group VII b:6.0 Gy + CdCl2 + Liv.52 AUTOPSY Fiveanimalsfromeachgroupwereautopsiedbycervicaldislocationateachpost-treatment interval of 1, 2, 4, 7, 14 and 28 days. The weight of animals was recorded before the autopsy. Five normal mice were also autopsied. Immediatelyaftertheautopsythebloodwascollectedbycardiacpunctureinheparinized tubes for various haematological studies. www.wjpps.com Vol 4, Issue 01, 2015.

1620 Sharma et al.World Journal of Pharmacy and Pharmaceutical Sciences HaematologicalParameters:Thevarioushaematologicalparametersestimatedwereas follows: Red blood corpuscles (R.B.C.) RED BLOOD CORPUSCLES (RBC) Thenumberofredbloodcorpusclesinthebloodwasestimatedbyvisualmethodusing improved double ruling Neubauer haemocytometer as given by plum (1936). Principle : The RBC number in 1 cu mm. being in lakhs makes the dilution of blood essential forvisualenumeration.Thisisdonebyusingisotonicdilutingfluid,whichalsoprevents haemolysis. Mercuric chloride in the fluid helps in fixing the cells. Reagents : The chemicals were dissolved in glass distilled water and final volume was made up to 200 ml. Procedure : Well-mixed whole blood was drawn to the 0.5 mark in a red cell diluting pipette anddilutedto101markwithR.B.C.dilutingfluid.Thepipettewasshakenforafew minutesandthefirstfewdropswerediscarded.Thecountingchambersofthe haemocytometer were charged with blood mixture from the pipette. The cells were allowed to settleinthechamberfor1 to2minutesandwere thencountedunder themicroscopeat the magnification of 675X. The count was made in the five of the 25 small squares of the central squares. Each small square has an area of 1/25sq. mm. Calculation : The average number was obtained by dividing the total count by five.Number of R.B.C. (Per cubic mm) = Average no x 200x25x10 RESULTS AND DISCUSSION RBC The changes in values of RBC (million/cubic mm.) in various groups are mentioned in table-1 and histogram-1. Group I (Sham-irradiated)The value of RBC in sham irradiated mice was 11.23 + 0.035. GroupII(CdCl2 ):AfallinthevalueofRBCwasnotedonday-1(9.76+0.027).This decreasingtrendcontinueduptoday-14(8.10+0.023)significantly(p